M Gautier

A Capitan

S Fritz

A Eggen

D Boichard

T Druet

Characterization of the DGAT1 K232A and Variable Number of Tandem Repeat Polymorphisms in French Dairy Cattle

Keywords: quantitative trait locus, dairy trait, acyl-CoA:diacylglycerol acyltransferase gene (DGAT1), variable number of tandem repeats

A quantitative trait locus (QTL) underlying different milk production traits has been identified with a high significance threshold value in the genomic region containing the acylCoA:diacylglycerol acyltransferase (DGAT1) gene, in the 3 main French dairy cattle breeds: French Holstein, Normande, and Montbe ´liarde. Previous studies have confirmed that the K232A polymorphism in DGAT1 is responsible for a major QTL underlying several milk production traits in Holstein dairy cattle and several other bovine breeds. In this study, we estimate the frequency of the 2 alternative alleles, K and A, of the K232A polymorphism in French Holstein, Normande, and Montbe ´liarde breeds. Although the K allele segregates in French Holstein and Normande breeds with a similar effect on production traits, the existence of additional mutations contributing to the observed QTL effect is strongly suggested in both breeds by the existence of sires heterozygous at the QTL but homozygous at the K232A polymorphism. One allele at a variable number of tandem repeats (VNTR) locus in the 5′ noncoding region of DGAT1 has been recently proposed as a putative causative variant. In our study, this marker was found to present a high mutation rate of 0.8% per gamete and per generation, making the allele diversity observed compatible with that expected under neutrality. Moreover, among the sires homozygous at the K232A polymorphism, no allele at the VNTR can fully explain their QTL status. Finally, no allele at the VNTR was found to be significantly associated with the fat percentage variation in the 3 breeds simultaneously after correction for the effect of the K232A polymorphism. Therefore, our results suggest the existence of at least one other causative polymorphism not yet described. Because the A allele is nearly fixed in the Montbe ´liarde breed, this breed represents an interesting model to identify and

INTRODUCTION

A QTL with a major influence on several milk production traits, and particularly on fat content, has been identified at the centromeric end of bovine chromosome 14 (BTA14; [START_REF] Coppieters | A QTL with major effect on milk yield and composition maps to bovine chromosome 14[END_REF][START_REF] Heyen | A genome scan for QTL influencing milk production and health traits in dairy cattle[END_REF][START_REF] Looft | A mammary gland EST showing linkage disequilibrium to a milk production QTL on bovine Chromosome 14[END_REF][START_REF] Boichard | Detection of genes influencing economic traits in three French dairy cattle breeds[END_REF]. Furthermore, fine mapping experiments have identified a nonconservative K232A polymorphism inside the acylCoA:diacylglycerol acyltransferase gene (DGAT1), which has been proposed as the causative mutation or quantitative trait nucleotide (QTN) explaining all the variation due to the QTL [START_REF] Grisart | Positional candidate cloning of a QTL in dairy cattle: Identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition[END_REF]. Additional studies have demonstrated the effect of this mutation on the function of the resulting enzyme, which plays a fundamental role in triacylglycerol metabolism [START_REF] Grisart | Genetic and functional confirmation of the causality of the DGAT1 K232A quantitative trait nucleotide in affecting milk yield and composition[END_REF].

Other studies have shown that the 2 alleles at the K232A polymorphism segregate in several breeds from different countries [START_REF] Kaupe | DGAT1 polymorphism in Bos indicus and Bos taurus cattle breeds[END_REF]. The K232A mutation probably occurred early in the history of cattle domestication or even before domestication [START_REF] Winter | Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content[END_REF][START_REF] Kaupe | DGAT1 polymorphism in Bos indicus and Bos taurus cattle breeds[END_REF]. Nevertheless, the estimated frequency of the wild-type lysine variant (the K allele) varies greatly according to the population considered, which might reflect different breeding objectives regarding milk composition in different countries and breeds. The effect of the mutation on milk production traits has been extensively characterized and the K allele is always associated with an increase in fat content, fat yield, and protein content, and a decrease in protein and milk yields.

Evidence for the existence of additional mutations contributing to the observed QTL effect has been reported recently [START_REF] Bennewitz | The DGAT1 K232A mutation is not solely responsible for the milk production quantitative trait locus on the bovine chromosome 14[END_REF]. In particular, one allele at a variable number of tandem repeat (VNTR) locus located in the 5′ noncoding region of DGAT1 has been Seventeen sires were included in the design as sons; thus, a total of 3,049 (3,025 + 24) individuals were used.

proposed as a putative causative variant [START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF].

In this paper, we report the characterization of both the VNTR and the K232A polymorphisms in the DGAT1 gene in the 3 main French dairy cattle breeds (Holstein, Normande, and Montbe ´liarde). We also investigate the existence of putative causative alleles at the VNTR locus.

MATERIALS AND METHODS

Experimental Data

Experimental Design. As detailed in Table 1, the animals considered in this study are organized according to 3 breed-specific granddaughter designs (GDD; [START_REF] Weller | Power of daughter and granddaughter designs for determining linkage between marker loci and quantitative trait loci in dairy cattle[END_REF]) including 6, 9, and 26 half-sib families for the Montbe ´liarde, Normande, and Holstein breeds, respectively. A total of 3,025 progeny-tested sons were used. In addition to the 14 initial families [START_REF] Boichard | Detection of genes influencing economic traits in three French dairy cattle breeds[END_REF], 27 new families (4, 6, and 17 in the Montbe ´liarde, Normande, and Holstein breeds, respectively) derived from the French marker-assisted selection (MAS) program [START_REF] Boichard | Implementation of marker-assisted selection in French dairy cattle[END_REF] were integrated in this design.

Daughter yield deviations (DYD) for 5 milk production traits (milk, fat, and protein yields and fat and protein percentages) and their corresponding reliabilities were obtained from the French national evaluation. DNA from all bulls had been extracted during the previously mentioned studies [START_REF] Boichard | Implementation of marker-assisted selection in French dairy cattle[END_REF][START_REF] Boichard | Detection of genes influencing economic traits in three French dairy cattle breeds[END_REF] and was available.

Genetic Markers. Genotypes for 2 bovine microsatellite markers CSSM066 and ILSTS039 were available from the MAS and initial QTL programs [START_REF] Boichard | Implementation of marker-assisted selection in French dairy cattle[END_REF][START_REF] Boichard | Detection of genes influencing economic traits in three French dairy cattle breeds[END_REF]. However, in the 2003 QTL program, animals had been genotyped only for CSSM066; thus, 2,941 (97%) and 1,514 (50%) sons were genotyped for CSSM066 and ILSTS039, respectively.

For the VNTR, the genotyping procedure consisted of a fluorescent PCR amplification with the following primers VNTR_F: TCAGGATCCAGAGGTACCAG and VNTR_R: CAATGAGAAGGCACGGACTGTGAA. For a Journal of Dairy Science Vol. 90 No. 6, 2007 5-repeat allele, the length of the resulting PCR product was 228 bp. The PCR reactions were performed using the Go-Taq Flexi (Promega, Paris, France) according to the manufacturer's recommendations on a PTC-100 thermocycler (MJ Research, Paris, France) in a final volume of 10 L. Samples were preheated for 5 min at 95°C, subjected to 40 cycles (95°C for 30 s, 58°C for 30 s, and 72°C for 30 s), and a final extension step of 5 min at 72°C. The resulting 228-bp PCR products were purified on a Sephadex G50 column (Amersham, Paris, France) before running on a Megabace 96 capillary sequencer (Molecular Dynamics, Paris, France). Raw data were then analyzed using Genetic Profiler v1.5 (Molecular Dynamics). Thereafter, alleles were named according to the number of repetitions (R) of the 18-bp motif. Some genotyped individuals were sequenced to confirm the expected number of repeats. In total, 2,958 (98%) progeny-tested sons were genotyped for this marker.

The K232A polymorphism was analyzed using the TaqMan allelic discrimination technology according to the standard genotyping protocol. The TaqMan probes (CCGTTGGCCGCCTT and CCGTTGGCCTTCTTA specific for the A and K alleles, respectively) and primers (DGAT1-F: CGCTTGCTCGTAGCTTTGG and DGAT1-R: CCGCGGTAGGTCAGGTTGT) were provided by Applied Biosystems (Paris, France). This procedure genotyped 3,020 (100%) progeny-tested sons for this marker.

For some nongenotyped sires from the GDD, marker genotypes were reconstructed based on the genotypes of their sons. This provided 24 additional genotyped individuals for the VNTR, the K232A polymorphism, and CSSM066, and 14 more for ILSTS039.

Pedigrees. Extended pedigrees were used for mixed model analyses. In addition to the genotyped animals, their ancestors over 3 generations were included in the pedigree. Noninformative ancestors; that is, ancestors with no parents and at most one progeny, were deleted iteratively. The size of these pedigree files was 1,006, 1,427, and 4,833 animals for the Montbe ´liarde, Normande, and Holstein breeds, respectively. The data files contained 422, 578, and 2,259 animals with records, respectively.

Statistical Analyses

Estimation of Allelic Frequencies. Estimation of allelic frequencies was done on the founder alleles. In the 3 different breed-specific GDD, these corresponded to the 2 sire alleles and each son's maternally inherited alleles.

Linkage Map Construction. Marker order and map distances were estimated using CRIMAP 2.4 software [START_REF] Green | Documentation for CRI-MAP, version 2.4[END_REF]. The CHROMPIC option was subsequently used to identify unlikely double crossovers. Final map distances were computed based on Haldane's mapping function.

QTL Mapping Analysis. Linkage analysis was performed using a regression interval analysis [START_REF] Haley | A simple regression method for mapping quantitative trait loci in line crosses using flanking markers[END_REF] with the web-based version of the QTL Express software [START_REF] Seaton | QTL Express: Mapping quantitative trait loci in simple and complex pedigrees[END_REF] fitting a 1-QTL model every centimorgan along the considered region. Records used were equal to twice the DYD of the bulls, weighted by their respective reliability. A 95% chromosome-wise significance threshold was computed based on 10,000 permutations. The P-value associated with the null hypothesis of a QTL homozygosity was calculated based on the t-test value at the maximum peak position. The rejection threshold was set to 5%.

VNTR and K232A Association Tests. Association tests were performed considering the 2 markers simultaneously with the following model:

y ij = s i + n kj b k + n vj b v + e ij
where y ij is the DYD of son j of sire i, s i is the fixed effect of sire i, n kj is the number of copies of the K allele carried by son j at the K232A polymorphism, n vj is the number of copies of the VNTR allele considered, b k and b v are, respectively, half the allele substitution effect of the K allele at the K232A polymorphism and the VNTR allele, and e ij is the residual effect associated to this record. The weight of each record was computed as already described [START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF]. Because more than 2 alleles were segregating for the VNTR, all alleles with a frequency greater than 0.05 were successively tested using the above model.

Estimation of the Effect of the K232A Polymorphism. To estimate the effect of the K232A polymorphism, the traits were modeled separately using the following linear mixed model:

y = + n k b k + Zu + e
where y is a vector containing twice the DYD for bulls, is a vector with the fixed effect of the mean, n k is a vector containing the number of copies of the K allele (0, 1, or 2) corresponding to the DYD, b k is the allele substitution effect, u is a vector of random polygenic effects, and e is a vector of random residual effects; Z is a design matrix relating the DYD of the individuals to their corresponding polygenic effect.

The (co)variance structure was:

var ⎡ ⎢ ⎣ u e ⎤ ⎥ ⎦ = ⎡ ⎢ ⎣ Aσ 2 a 0 0 Rσ 2 e ⎤ ⎥ ⎦
where σ 2 a is the variance associated to the random polygenic effect and σ 2 e the residual variance; A is the additive relationship matrix and R is a diagonal matrix containing the inverse of the weight of the corresponding DYD. These weights were daughter equivalents and were estimated as previously described [START_REF] Vanraden | Derivation, calculation, and use of national animal model information[END_REF] with a correction for the number of cows per herd. These analyses were performed using the ASREML package [START_REF] Gilmour | ASREML reference manual[END_REF].

Haplotype Analysis and Linkage Disequilibrium Estimation. The most likely haplotype phases of each individual from the different breed-specific pedigrees were computed using the deterministic algorithm already described [START_REF] Boichard | Detection of genes influencing economic traits in three French dairy cattle breeds[END_REF]. Estimation of haplotype frequencies and linkage disequilibrium (LD) was performed on the founder haplotypes (see above).

Pairwise LD measures were derived from the standard measure of LD between 2 alleles at 2 different loci: D ij = p(A i B j )p(A i )p(B j ), where p(A i ) is the frequency of allele A i at locus A, p(B j ) the frequency of allele B j at locus B and p(A i B j ) the frequency of haplotype A i B j as estimated from the population. Three multiallelic LD measures derived from this standard measure were considered in the study.

First, multiallelic D′ was measured as [START_REF] Hedrick | Gametic disequilibrium measures: Proceed with caution[END_REF]:

D′ = ∑ k i=1 ∑ 1 j=1 p(A i )p(B j ) ⎢ ⎢ ⎢ D ij D max ij ⎢ ⎢ ⎢
,

where k and l are the number of alleles for marker A and B, respectively, and

D max ij = min{p(A i )p(B j ), [1 -p(A i )][1 -p(B j )]} when D ij < 0, D max ij = min{p(A i )[1 -p(B j )], p(B j )[1 -p(A i )]} when D ij ≥ 0.
Second, multiallelic r 2 was measured as [START_REF] Hill | Linkage disequilibrium in finite populations[END_REF])

r 2 = ∑ k i=1 ∑ 1 j=1 D 2 ij [1 -p(A i )][1 -p(B j )]
.

Finally, the multiallelic χ 2 statistic is defined as [START_REF] Hill | Linkage disequilibrium among multiple neutral alleles produced by mutation in finite population[END_REF])

χ 2 = N ∑ k i=1 ∑ 1 j=1 D 2 ij p(A i )p(B j ) ,
where N is the number of haplotypes considered to compute the corresponding pairwise measure. χ 2 is asymp- totically distributed according to a χ 2 distribution with (k -1)(l -1) degrees of freedom. The threshold for significance of LD was set to 0.001.

To limit bias introduced by rare alleles in the estimation of the different measures and to preserve power to reject the null hypothesis of absence of LD, marker alleles with frequency <0.05 were pooled.

RESULTS

Characterization of the K232A and VNTR Polymorphisms

The lysine variant (K allele) at the K232A polymorphism was present in the 3 breeds and its frequency ranged from 0.04 in the Montbe ´liarde breed to 0.13 in the Normande breed and 0.37 in the Holstein breed (Table 2). Distributions of genotypes are presented in Table 2 for the 3 breeds. Only 14 out of the 384 Montbe ´liarde bulls were heterozygous for K232A and none was homozygous for the lysine variant (KK). Indeed, most animals were homozygous for the alanine variant (AA) in both the Montbe ´liarde and Normande breeds. In the Holstein breed, where the K allele is much more frequent, approximately 10% of the bulls were homozygous (KK) and 43% were heterozygous.

Seven different alleles (from 2 to 8 repetitions of the 18-bp motif) at the VNTR were identified in the present study with corresponding frequencies varying according to breed (Table 3). The 2R allele segregated both in the Montbe ´liarde and Normande breeds and was not reported in previous studies [START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF][START_REF] Sanders | Characterization of the DGAT1 mutations and the CSN1S1 promoter in the German Angeln dairy cattle population[END_REF]. For Holsteins, the VNTR allele frequencies varied within a similar range in Germany and France (Table 3).

Map Construction and Estimation of the VNTR Mutation Rate

When considering all families simultaneously, 1,803, 472, 951, and 1,849 informative meioses were available for the VNTR, K232A, ILSTS039, and CSSM066 loci, respectively, to build a genetic map. This order was found to be the most likely and is in agreement with previous studies. Nevertheless, several inconsistencies appeared Journal of Dairy Science Vol. 90 No. 6, 2007 in the segregation of the VNTR when analyzing genotyping raw data. In particular, genotypes of 4 sons descending from 3 different homozygous sires (with 5R/5R genotypes) were not compatible for this marker. Filiations of these sons were unambiguously assessed based on genotyping data from other projects (data not shown). A total of 506 genotyped sons descended from a homozygous sire at the VNTR. Assuming the absence of genotyping errors, the estimated mutation rate for the VNTR could be set to 0.8% (4/506) per paternal gamete. This value was consistent with known features for a VNTRtype locus in other mammalian species [START_REF] Bois | Minisatellite instability and germline mutation[END_REF]. Additionally, based on further haplotype analyses, 3 mutational events out of the 4 identified were characterized and corresponded to repetition gains (from 5R to 6R).

The estimated 2-point Haldane genetic distances for each pair of markers are given in Table 4. It is worth noting that the VNTR and K232A polymorphisms were less than 10 kb apart. Thus, distances appeared strongly inflated for markers linked to the VNTR. Respectively, 1 recombinant out of 289 coinformative meioses for the VNTR/K232A marker pair was observed, 16 out of 522 for the VNTR/ILSTS039 marker pair, and 114 out of 1,155 for the VNTR/CSSM066 marker pair (131 in total). A small fraction of these recombinant gametes might have been created by mutational events. Only paternal gametes are informative in our design. Consequently, a mutation can create a recombinant gamete only if the maternally inherited allele is identical in state to one of the paternal alleles, or if the mutated allele is identical in state to the alternative paternal allele. Therefore, the observed inflation in genetic distances was probably the result of a recombination hot spot in this centromeric region and an underestimation of genetic distances when considering the K232A polymorphism due to a lack of informative meioses.

QTL Analysis. The QTL was detected in the Holstein breed for the 5 traits (P < 10 -4 ) whereas in the Montbe ´liarde and Normande breeds, the QTL was significant only for fat percentage (P < 10 -4 ). The heterozygous status at the QTL was determined using fat percentage as a reference in the 3 breeds, because this trait shows the most pronounced QTL effect. As shown in Table 5, all the Montbe ´liarde sires were homozygous AA at the K232A polymorphism and 5 were heterozygous at the QTL. In the Normande breed, only sire #1611 (out of the 3 sires that were heterozygous at the QTL) was also heterozygous at the K232A polymorphism. In the Holstein breed, the 11 sires heterozygous at the K232A polymorphism were also heterozygous at the QTL and 2 sires homozygous at the mutation were heterozygous at the QTL.

As previously reported [START_REF] Bennewitz | The DGAT1 K232A mutation is not solely responsible for the milk production quantitative trait locus on the bovine chromosome 14[END_REF], the existence of sires heterozygous at the QTL and homozy- The names of the alleles are given according to the number of repetitions (R) of the 18-bp motif.

2

From [START_REF] Sanders | Characterization of the DGAT1 mutations and the CSN1S1 promoter in the German Angeln dairy cattle population[END_REF]; the names of the alleles in this publication are indicated in parentheses.

3

From [START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF]; the names of the alleles in this publication are indicated in parentheses. Allele frequencies from this study correspond to those estimated on the basis of maternally inherited haplotypes (individuals belonging to a granddaughter design).

gous at the K232A polymorphism indicates that this latter mutation is not solely responsible for the QTL variation. The VNTR has been proposed as a putative candidate locus. In the Holstein and Normande breeds, the 4 sires (in Normande: #1406 and #1788; in Holstein: #3498 and #3501) homozygous AA at the K232A polymorphism and heterozygous at the QTL have the 4R allele at the VNTR in common. Nevertheless, 4 Holstein sires (#3442, #3480, #3481, and #6145) and 3 Normande sires (#1961, #2409, and #2618) homozygous (AA) at the K232A polymorphism carried 1 copy of this same allele at the VNTR but were homozygous at the QTL. In the Montbe ´liarde breed, among the 5 sires homozygous (AA) at the K232A polymorphism and heterozygous at the QTL, 4 (#1298, #1312, #1368, and #1531) carried 1 copy of the 5R and the 4R allele at the VNTR and the other sire (#1103) was homozygous for the 5R allele at the VNTR. The 7R allele, which was proposed as having the most pronounced effect in German Holsteins (Kuhn et al., 2004) and in the Angeln breed [START_REF] Sanders | Characterization of the DGAT1 mutations and the CSN1S1 promoter in the German Angeln dairy cattle population[END_REF], was not carried by any Normande and Montbe ´liarde sires. Out of the 6 Holstein sires (#3501, #3442, #3480, #3500, #3514, #4729, and #6865) homozygous at the K232A polymorphism and carrying 1 copy of this allele at the VNTR, only sire #3501 was heterozygous at the QTL.

VNTR and K232A Association Tests. Both the VNTR and K232A polymorphisms were previously suggested as causative. Thus, we performed association tests considering the 2 markers simultaneously to test the effect of the different VNTR alleles after correction for the K232A effect. As expected, the allele substitution effect for the K232A polymorphism was highly significant (P < 10 -4 ) for fat content in the 3 breeds. In the Montbe ´liarde breed it was also significant (P < 0.05) for milk yield and protein content. In the Normande and Holstein breeds, the effect was significant (P < 10 -4 ) for all traits except for protein yield in the Normande breed.

No allele at the VNTR was associated with trait variation across all 3 breeds. Nevertheless, in the Montbe ´liarde breed, the 4R allele was associated with milk and protein yield (P < 0.01), fat content (P < 10 -4 ), and protein content (P < 0.05) variations, whereas in the Normande breed, only the 3R allele was associated with protein yield variation (P < 0.05). Finally, as observed in the German Holstein population [START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF], the 7R allele in the French Holstein breed was significantly associated with an increase in fat yield (P < 0.05), protein content (P < 0.05), and fat content (P < 10 -4 ).

Characterization of the Effect of the K232A Polymorphism. The average allelic substitution effect of the K232A polymorphism for dairy traits is presented in Table 6. The allelic substitution effect was estimated using twice the DYD. Therefore, it indicates the difference in terms of EBV and not of PTA. In the Holstein breed, the K allele increased fat yield (+14.90 kg) and fat (+0.34 %) and protein (+0.08%) contents, whereas it decreased milk (-351.2 kg) and protein (-4.6 kg) yields. These results are consistent for the 3 breeds but the magnitude of substitution effects varied. These estimated average allelic substitution effects were in agreement with previous studies [START_REF] Grisart | Positional candidate cloning of a QTL in dairy cattle: Identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition[END_REF][START_REF] Spelman | Characterization of the DGAT1 gene in the New Zealand dairy population[END_REF][START_REF] Thaller | Effects of DGAT1 variants on milk production traits in German cattle breeds[END_REF][START_REF] Weller | Population-wide analysis of a QTL affecting milk-fat production in the Israeli Holstein population[END_REF].

The variance due to the QTL can be estimated as 2p(1p)a 2 , where a is the additive effect and p is the frequency of one allele. The variances obtained with this approximation are presented for the Holstein breed, expressed as a percentage of the total genetic variance, in Table 6. The variances range from 2.4 to 42.5% of the total genetic variance for protein yield and fat content, respectively.

Haplotype Analysis and D′ Among Markers. As shown in Table 7, all markers were found to be in significant LD with each other among the 3 different populations considered. As expected, irrespective of the multiallelic measure considered (r 2 or D′), LD decreases when the genetic distance separating the markers increases. Interestingly, r 2 values for the 3 marker pairs involving the K232A polymorphism are all far from 1, indicating Journal of Dairy Science Vol. 90 No. 6, 2007 that no allele at the 3 other markers (even the VNTR located only 10 kb away) behaved as a perfect surrogate for the K232A alleles. For instance, as shown in Table 8, in the Montbe ´liarde breed the K allele at the K232A polymorphism was always associated with the 5R allele at the VNTR (the D′ value is thus equal to 1), but an almost equal fraction of this latter allele at the VNTR was associated with the A allele at the K232A polymorphism (the r 2 value is thus far from 1). Similarly, in French Holsteins, as previously observed in German Holsteins [START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF], the K allele was almost always associated with the 5R allele at the VNTR (D′ = 0.98). In the Normande breed, the K allele was associated mainly with the 4R allele at the VNTR, whereas a much smaller fraction was also associated with the 5R allele (D′ = 0.68).

DISCUSSION

In our study, a QTL underlying fat percentage was detected in the 3 main French dairy cattle breeds in the region containing DGAT1. Thus, this region harbors one or several genes affecting this trait. In the Holstein breed, the K232A polymorphism appeared to contribute to a large part of the genetic variation due to the QTL. All sires heterozygous at the K232A polymorphism were also heterozygous at the QTL. The frequency of the K allele estimated in our Holstein population is in the range reported in other studies: 0.40 in New Zealand [START_REF] Spelman | Characterization of the DGAT1 gene in the New Zealand dairy population[END_REF], 0.35 [START_REF] Winter | Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content[END_REF] or 0.55 [START_REF] Thaller | Effects of DGAT1 variants on milk production traits in German cattle breeds[END_REF] in Germany, 0.16 in Israel [START_REF] Weller | Population-wide analysis of a QTL affecting milk-fat production in the Israeli Holstein population[END_REF], and 0.27 in Brazil [START_REF] Lacorte | DGAT1 K232A polymorphism in Brazilian cattle breeds[END_REF].

The effects of the K232A polymorphism were estimated on an EBV scale but with twice the DYD as records (which were not regressed as EBV). For the Holstein breed, the results were close to those previously published [START_REF] Grisart | Positional candidate cloning of a QTL in dairy cattle: Identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition[END_REF][START_REF] Spelman | Characterization of the DGAT1 gene in the New Zealand dairy population[END_REF][START_REF] Thaller | Effects of DGAT1 variants on milk production traits in German cattle breeds[END_REF], which were computed on a PTA scale with DYD as records. The K allele improved fat yield and fat and protein contents and had a negative effect on the other dairy traits. The effects in the 3 breeds were consistent with respect to their direction but the magnitude of the estimated effects changed. Similar tendencies have been described in Fleckvieh and Braunvieh cattle and Ayrshire and Jersey breeds, respectively [START_REF] Spelman | Characterization of the DGAT1 gene in the New Zealand dairy population[END_REF][START_REF] Thaller | Effects of DGAT1 variants on milk production traits in German cattle breeds[END_REF]. However, because the K allele is rare in the Montbe ´liarde and Normande breeds, the standard error of the estimators of these effects is large in these breeds.

The combination of allele frequency and allele substitution effects gives an indication on genetic variance due to this polymorphism. As expected, this variance was very large for fat content in the Holstein breed (more than 40% of the genetic variation). The proportion of explained genetic variance estimated in this study for the different traits was close to that originally reported [START_REF] Grisart | Positional candidate cloning of a QTL in dairy cattle: Identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition[END_REF]. In addition, using a variance component approach on a larger sample of the French Holstein population, the proportion of genetic variance explained by the QTL traced by closely linked markers has recently been found to be very similar [START_REF] Druet | Estimation of genetic parameters for quantitative trait loci for dairy traits in the French Holstein population[END_REF]. Nevertheless, because of a lower frequency of the K allele in the Israeli Holstein population, lower values have been observed [START_REF] Weller | Population-wide analysis of a QTL affecting milk-fat production in the Israeli Holstein population[END_REF]. Interestingly, the estimated variance for protein yield was relatively small, indicating that the use of DGAT1 to improve this The number of reconstructed haplotypes containing information for both markers in the considered population. Although there is no doubt that the K232A polymorphism had a strong effect, at least in the Holstein breed, it seemed not solely responsible for the QTL as previously postulated [START_REF] Bennewitz | The DGAT1 K232A mutation is not solely responsible for the milk production quantitative trait locus on the bovine chromosome 14[END_REF][START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF]. Indeed, 2 Holstein sires appeared to be heterozygous at the QTL but were not heterozygous at the K232A polymorphism. A similar trend was observed in the Normande breed. Moreover, in the Montbe ´liarde breed, the K232A polymorphism was clearly not the underlying QTN. These results confirm that another mutation must contribute to the observed QTL effect. Recently, a VNTR in the 5′ noncoding region of DGAT1 has been proposed to explain the additional part of the QTL variance, and one allele (7R in our study) was suggested as a putative causal allele [START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF][START_REF] Sanders | Characterization of the DGAT1 mutations and the CSN1S1 promoter in the German Angeln dairy cattle population[END_REF].

Our study showed little evidence for this hypothesis. Indeed, among the 6 Holstein sires homozygous at the K232A polymorphism and carrying 1 copy of the 7R allele, only one was heterozygous at the QTL. In the Normande and Montbe ´liarde breeds, this allele was only present at a low frequency (<0.005) and its effect cannot be estimated. Recently, a functional analysis [START_REF] Furbass | Alleles of the bovine DGAT1 variable number of tandem repeat associated with a milk fat QTL at chromosome 14 can stimulate gene expression[END_REF] established that the Sp1 transcription factor could bind to the VNTR and demonstrated experimentally its functional relevance regarding induction of the transduction of a reporter gene. Nevertheless, no significant difference in the stimulating effect of the 3 different alleles compared (3R, 6R, and 7R) was observed. These different elements do not support a direct effect of the VNTR. For each breed considered, a different allele (3R in Normande breed, 4R in Montbe ´liarde breed, and 7R in Holstein breed) was found to be associated with a significant effect after correction for the K232A effect. This association of some VNTR alleles may be the result of residual LD existing with at least one additional causal polymorphism other than the K232A polymorphism underlying the QTL. Yet, a comprehensive screen for mutations in the DGAT1 gene (in particular in the 5′ noncoding region) did not identify any significant association with the trait for any of the polymorphisms found except the VNTR [START_REF] Winter | Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content[END_REF][START_REF] Kuhn | Evidence for multiple alleles at the DGAT1 locus better explains a quantitative trait locus with major effect on milk fat content in cattle[END_REF]; therefore, the additional causal variants might have to be searched in the surrounding intergenic regions. The mutational event creating the K232A polymorphism from the K allele (the ancestral state) probably occurred early in the history of domestication of European cattle [START_REF] Winter | Association of a lysine-232/alanine polymorphism in a bovine gene encoding acyl-CoA:diacylglycerol acyltransferase (DGAT1) with variation at a quantitative trait locus for milk fat content[END_REF] more than 10,000 yr ago. In our study, the most important allelic diversity at the ILSTS039 microsatellite and the VNTR markers Journal of Dairy Science Vol. 90 No. 6, 2007 was associated with the A allele at the K232A polymorphism. The high mutation rate at the VNTR (0.8% per generation and paternal gamete from our study) and microsatellite loci (known to range from 10 -3 to 10 -4 ) can be considered as the primary factor generating allelic diversity in the haplotypes carrying the A allele, given that the expected numbers of alleles observed at these 2 markers was in almost perfect agreement with a stepwise mutation model [START_REF] Ohta | A model of mutation appropriate to estimate the number of electrophoretically detectable alleles in a finite population[END_REF].

Additionally, the frequency of the ancestral K allele at the K232A polymorphism has increased only recently, particularly in the Holstein breed, possibly due to more pronounced selection favoring fat content. This might have contributed to markedly reduced allelic diversity at surrounding markers by a genetic hitchhiking phenomenon [START_REF] Grisart | Genetic and functional confirmation of the causality of the DGAT1 K232A quantitative trait nucleotide in affecting milk yield and composition[END_REF]. Alternatively, local and recent introgression of the K allele in some European breeds might explain the low level of within-breed diversity associated with the K-carrying haplotypes [START_REF] Kaupe | DGAT1 polymorphism in Bos indicus and Bos taurus cattle breeds[END_REF]. Indeed, some ancestral K alleles, probably from different origins, still segregate in the different cattle populations. In our study for instance, within the Normande and Holstein populations, the haplotypes carrying the K allele were mainly associated with different alleles at both the ILSTS039 and VNTR markers. On the other hand, the few K alleles segregating in the Montbe ´liarde breed might have been introduced recently during the known limited crossbreeding events with Red Holstein, which occurred in the 1970s. Indeed, in the Montbe ´liarde breed, the K alleles were all associated with the same ILSTS039 and VNTR alleles (218 and 5R, respectively) whereas in the Holstein breed, 85 and 99% of the K alleles were associated with the same 2 alleles.

CONCLUSIONS

The ancestral K allele of the DGAT1 K232A polymorphism was present in the 3 breeds studied, whereas the alternate A allele is nearly fixed in the Montbe ´liarde breed. In the Normande and French Holstein breeds, the effects of the allele were consistent with a large effect on milk production and composition traits. The VNTR polymorphism explained only a small fraction of the variance of the QTL (after correction for the K232A effect). Other uncharacterized polymorphism(s) certainly exist at the centromeric end of bovine chromosome 14 because it has been shown that the K232A polymorphism is not solely responsible for the QTL variation. In the Montbe ´liarde breed, all the heterozygous sires at the QTL were homozygous at the K232A polymorphism. This breed represents an excellent model to identify additional mutations contributing to the observed QTL effect.

2

  The number of different haplotypes exiting in the considered population.Journal of Dairy Science Vol. 90 No. 6, 2007 trait (the most important in the French breeding goal) is less efficient.

Table 1 .

 1 Description of the 3 breed-specific granddaughter designs used in the study

		Number	Average	Number of
		of families	family	progeny-tested
	Breed	(sires)	size	sons
	Montbe ´liarde	6	65 (41 to 98)	387
	Normande	9	60 (38 to 74)	541
	Holstein	26	81 (34 to 231)	2,097
	All	41 1	74 (34 to 231)	3,025
	1

Table 2 .

 2 Frequency of the K allele and genotypes of animals with records in the 3 breeds

		Frequency		Genotype
	Breed	of allele K	KK	AK	AA
	Montbe ´liarde	0.040	0	14	370
	Normande	0.130	11	122	402
	Holstein	0.369	177	944	965

Table 3 .

 3 Allele frequencies of the variable number of tandem repeat polymorphism (VNTR) in the 3 breeds and comparisons with previous reports

	Frequency

Table 4 .

 4 Two-point and multipoint (in parentheses) Haldane genetic distances (cM) for the different marker pairs

	Marker
	name	K232A	ILSTS039	CSSM066
	VNTR	0.035 (0.035)	3.16 (2.61)	11.0 (10.2)
	K232A		1.61 (2.57)	8.82 (10.1)
	ILSTS039			6.96 (7.54)

Journal of Dairy Science Vol. 90

No. 6, 2007

Table 5 .

 5 Results for the within-family QTL analysis for milk fat

	percentage trait
				VNTR_DGAT	t-value for
	Breed	Family	K232A	haplotype	fat yield
	Montbe ´liarde	1103	A/A	5R_A/5R_A	2.15*
		1298	A/A	5R_A/4R_A	2.63**
		1312	A/A	5R_A/4R_A	3.25**
		1368	A/A	5R_A/4R_A	2.01*
		1531	A/A	5R_A/4R_A	3.23**
		1736	A/A	4R_A/4R_A	0.33
	Normande	1406	A/A	4R_A/3R_A	2.05*
		1788	A/A	4R_A/6R_A	3.48**
		1611	K/A	4R_A/4R_K	4.37**
		1528	K/K	5R_K/4R_K	0.23
		1537	A/A	3R_A/6R_A	0.43
		1855	A/A	4R_A/4R_A	0.2
		1961	A/A	4R_A/3R_A	0.88
		2409	A/A	4R_A/3R_A	1.59
		2618	A/A	4R_A/3R_A	0.39
	Holstein	3498	A/A	4R_A/6R_A	2.08*
		3501	A/A	4R_A/7R_A	4.7**
		2108	K/A	5R_K/7R_A	4.53**
		6146	K/A	5R_K/7R_A	6.69**
		3465	K/A	5R_K/4R_A	5.11**
		3468	K/A	5R_K/3R_A	8.07**
		3487	K/A	5R_K/6R_A	8.08**
		3492	K/A	5R_K/4R_A	3.98**
		3502	K/A	5R_K/6R_A	9.87**
		4442	K/A	5R_K/4R_A	6.42**
		5245	K/A	5R_K/5R_A	5.84**
		5970	K/A	5R_K/6R_A	6.85**
		5974	K/A	5R_K/5R_A	7.32**
		3442	A/A	7R_A/4R_A	1.86
		3480	A/A	7R_A/4R_A	0.93
		3500	A/A	7R_A/6R_A	0.56
		3514	A/A	7R_A/6R_A	0.51
		4729	A/A	7R_A/5R_A	0.19
		3481	A/A	4R_A/6R_A	0.46
		5972	A/A	5R_A/6R_A	1.79
		6109	A/A	4R_A/4R_A	1.49
		6143	A/A	5R_A/3R_A	1.85
		6145	A/A	4R_A/3R_A	0.27
		6151	A/A	5R_A/6R_A	0.74
		6865	A/A	6R_A/6R_A	-1
		7210	K/K	5R_K/5R_K	-
	1 Sires are homozygous for the 4 markers and thus could not be
	analyzed.

*P < 0.05; **P < 0.01.

Table 6 .

 6 Estimates of the average substitution effects on 5 production traits in the 3 different breeds from the regression of individual daughter yield deviations on the number of K alleles inherited in the 3 breeds

	Trait	Montbe ´liarde	Normande	Holstein
	Milk yield
	Average substitution effect, kg (SE) Proportion of genetic variance explained, 1 %	-611 (170) N A 2	-142 (61) NA	-351 (22) 13.1
	Fat yield
	Average substitution effect, kg (SE)	6.24 (6.5)	9.30 (2.5)	14.9 (0.88)
	Proportion of genetic variance explained, 1 %	NA	NA	14.8
	Protein yield
	Average substitution effect, kg (SE) Proportion of genetic variance explained, 1 %	-14.3 (5.3) NA	-0.610 (2.0) NA	-4.63 (0.66) 2.40
	Fat content
	Average substitution effect, ‰ (SE)	4.25 (0.51)	2.54 (0.26)	3.43 (0.090)
	Proportion of genetic variance explained, 1 %	NA	NA	42.5
	Protein content
	Average substitution effect, ‰ (SE)	0.960 (0.32)	0.690 (0.14)	0.760 (0.050)
	Proportion of genetic variance explained, 1 %	NA	NA	13.0
	1 Estimates of the proportion of genetic variance due to the K232A polymorphism have only been computed
	in the Holstein breed (see text).
	2 NA = not analyzed.

Table 7 .

 7 Linkage disequilibrium of marker pairs within the different breeds

				Sample Haplotype
	Marker pair	Breed	df	size 1	number 2	D′	r 2	χ 2	P
	VNTR/K232A VNTR/ILSTS039 VNTR/CSSM066 K232A/ILSTS039 K232A/CSSM066 ILSTS039/CSSM066 Holstein Holstein Montbe ´liarde Normande Holstein Montbe ´liarde 15 4 3 4 16 Normande 16 Holstein 20 Montbe ´liarde 15 Normande 20 Holstein 4 Montbe ´liarde 5 Normande 4 Holstein 5 Montbe ´liarde 5 Normande 5 20 Montbe ´liarde 25 Normande 20	1,767 379 420 838 245 262 1,783 373 417 821 251 253 1,814 379 408 846 245 250	8 5 9 23 17 18 30 20 24 10 7 9 12 8 12 29 26 22	0.98 0.269 1.0 0.042 0.68 0.049 0.49 0.065 0.38 0.039 0.61 0.122 0.42 0.035 1,057 752 23.1 4 × 10 -05 2 × 10 -161 49.3 5 × 10 -10 570 5 × 10 -111 66.9 2 × 10 -08 170 7 × 10 -28 4 × 10 -211 0.30 0.016 75.0 6 × 10 -10 0.36 0.026 164 2 × 10 -24 0.70 0.174 269 6 × 10 -57 1.0 0.020 29.5 2 × 10 -05 0.74 0.067 48.7 7 × 10 -10 0.45 0.075 337 1 × 10 -70 0.93 0.041 70.2 9 × 10 -14 0.24 0.008 25.5 1 × 10 -04 0.55 0.096 585 4 × 10 -111 0.40 0.027 218 9 × 10 -33 0.31 0.016 119 5 × 10 -16
	1

Table 8 .

 8 Population allele frequencies of the variable number of tandem repeat (VNTR)/K232A haplotypes

			Population frequency
	VNTR/K232A	German
	haplotype	Holstein 1 Holstein Normande Montbe ´liarde
	3R_A	0.021	0.041	0.249	0.000
	4R_A	0.176	0.160	0.307	0.456
	4R_K	0.006	0.002	0.101	0.000
	5R_A	0.167	0.203	0.118	0.393
	5R_K	0.207	0.350	0.025	0.045
	6R_A	0.218	0.128	0.198	0.095
	6R_K	0.000	0.001	0.002	0.000
	7R_A	0.207	0.115	0.000	0.011
	1 From Kuhn et al. (2004).

ACKNOWLEDGMENTS

We thank He ´le `ne Legros (LABOGENA) and Catherine Denis (INRA) for their help in marker genotyping and He ´le `ne Hayes (INRA) for English correction of the manuscript. We also thank the 2 anonymous reviewers for their helpful suggestions and corrections.


Caroline C Lacroux

Thi Hai

Chi Nguyen

Olivier Andréoletti

Françoise Prevot Prévot

Christelle C Grisez

Jean-Paul Bergeaud

Lucas Gruner

Jean-Claude Brunel

Dominique Francois

Philippe Dorchies

Olivier Andreoletti

Françoise Prevot

Philippe Jacquiet
email: p.jacquiet@envt.fr

Haemonchus contortus (Nematoda : Trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Haemonchus contortus is a common nematode parasite of small ruminants that causes important losses of milk, meat and wool production and death in young animals [START_REF] Tembely | The epidemiology of nematode infections in sheep in a cool tropical environment[END_REF][START_REF] Waller | The epidemiology of abomasal nematodes of sheep in Sweden, with particular reference to over-winter survival strategies[END_REF]. Due to the emergence of anthelmintic resistance [START_REF] Sangster | Anthelmintic resistance: past, present and future[END_REF][START_REF] Wolstenholme | Drug resistance in veterinary helminths[END_REF] and public concern about chemical residues in animal products, alternative control strategies are needed. Vaccination and genetic resistance of hosts have been viewed as sustainable methods of parasite control. Substantial progress and results have been obtained by using several H. contortus antigens, such as native H11 and H-gal-GP, to stimulate efficient levels of protective immunity in sheep [START_REF] Knox | The nature and prospects for gut membrane proteins as vaccine candidates for Haemonchus contortus and other ruminant trichostrongyloids[END_REF]. Nevertheless, no commercial vaccine is yet available. Between and within breed differences in host resistance are currently reported throughout the world [START_REF] Miller | Epidemiology of gastrointestinal nematode parasitism in Suffolk and Gulf Coast Native sheep with special emphasis on relative susceptibility to Haemonchus contortus infection[END_REF][START_REF] Woolaston | Prospects of breeding small ruminants for resistance to internal parasites[END_REF] but the mechanisms underlying this resistance are not well known. The immune response against gastrointestinal nematodes has been extensively studied in rodents infected with Trichinella spiralis, Nippostrongylus brasiliensis, Heligmosomoïdes polygyrus and Trichuris muris. Protective immunity against these parasites is largely dependant on the activation of CD4+ T helper cells and on the Th 2 polarization of the immune response [START_REF] Urban | The importance of Th2 cytokines in protective immunity to nematodes[END_REF], while a Th 1 response is associated with the survival of these parasites and increased host susceptibility to infection [START_REF] Else | Intestinal nematode parasites, cytokines and effector mechanisms[END_REF][START_REF] Finkelman | Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models[END_REF]. In humans, the protective immune response observed in natural Ascaris lumbricoïdes or Necator americanus infections is similarly associated with a high Th 2 -type cytokine polarization [START_REF] Cooper | Human infection with Ascaris lumbricoides is associated with a polarized cytokine response[END_REF][START_REF] Quinnell | Immune responses in human necatoriasis: association between interleukin-5 responses and resistance to reinfection[END_REF]. In ruminants, the nature of the immune response towards gastrointestinal helminths is less clear and the existence of a true Th 1 /Th 2 dichotomy was questioned in the late nineties [START_REF] Brown | Bovine type 1 and type 2 responses[END_REF][START_REF] Gill | Induction of T helper 1-and T helper 2type immune responses during Haemonchus contortus infection in sheep[END_REF][START_REF] Pernthaner | Cytokine mRNA and IFN-γ production in nematode resistant and susceptible line lambs artificially infected with gastrointestinal nematodes[END_REF]. Few studies on the polarization of the adaptive immune response in H. contortus infected sheep are available and the results have failed to prove an unequivocal Th 2 response [START_REF] Schallig | Immunological responses of sheep to Haemonchus contortus[END_REF]. Nevertheless, a Th 2 polarization has been recently dem-onstrated in Trichostrongylus colubriformis infections in sheep [START_REF] Pernthaner | Increased expression of interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha genes in intestinal lymph cells of sheep selected for enhanced resistance to nematodes during infection with Trichostrongylus colubriformis[END_REF].

In rodent models, Th 2 immune responses are almost invariably accompanied by local and systemic eosinophilia and mucosal mast cell hyperplasia, but also with local and general specific antibody responses (mainly IgA, IgG1 and IgE) [START_REF] Onah | Mucosal immunity against parasitic gastrointestinal nematodes[END_REF]. The same features are also encountered in sheep infected with H. contortus [START_REF] Schallig | Immunological responses of sheep to Haemonchus contortus[END_REF], Teladorsagia circumcincta [START_REF] Stear | Regulation of egg production, worm burden, worm length and worm fecundity by host responses in sheep infected with Ostertagia circumcincta[END_REF] and Trichostrongylus colubriformis [START_REF] Bendixsen | The sensitization of mucosal mast cells during infections with Trichostrongylus colubriformis or Haemonchus contortus in sheep[END_REF]. The impact of these effectors and their relative importance in host protection are still unclear. Moreover, sequences of the immune response and mobilization of the antibody and cellular effectors have not been systematically characterized in H. contortus infections. Therefore, the objectives of this study were (i) to define, using a quantitative reverse transcriptase (RT) PCR, the polarization of the immune response in lambs infected with H. contortus, (ii) to investigate the kinetics of Th 2 -type-associated effectors in the abomasal mucosa and (iii) to characterize the effects of this immune response on the nematode populations within the host.

MATERIALS AND METHODS

Experimental design

Animals

Eighty-one 3-month old female INRA 401 lambs were randomly allocated into three experimental groups: Group A: previously-infected, anthelmintic-treated and then challenged sheep (n = 29), Group B: primary-infected sheep (n = 28), and Group C: uninfected control sheep (n = 24). All sheep were born, reared and maintained worm-free in three separate pens before the experiments. Limited infections with coccidia occurred in all groups despite diclazuril treatments (Vecoxan, Janssen-Cilag, Issy-les-Moulineaux, France, 1 mg/kg BW) before the commencement of the experiment, but no clinical signs of coccidiosis were observed. Experimental lambs received granulated feed adapted to their age, as well as forage and tap water ad libitum. The animals were reared following European Union recommendations for animal welfare and under the supervision of the local INRA ethics committee.

Experimental infections and necropsies

Lambs of group A (previously-infected) were orally infected with 10 000 H. contortus third-stage larvae (L 3 ) of the Humeau strain (kindly provided by L. Gruner, INRA BASE, Tours-Nouzilly, France) 35 days before challenge. All lambs were treated with an oral administration of Levamisole (7.5 mg/kg) at day -7. The lambs of groups A (previously-infected) and B (primaryinfected) were orally infected with 10 000 H. contortus L 3 at D 0 . Six animals of each group were humanely killed at each of days 3, 7 and 15 post challenge (dpc) by exsanguination following intravenous injection of 10 mg/kg pentobarbital sodium. At day 28, eleven lambs from group A, ten from group B and six from group C were killed (Tab. I). Necropsy days were chosen to conform with the expected development of the parasite within the host: only early fourthstage larvae (L 4 ) at 3 dpc, majority of late L 4 at 7 dpc, immature worms at 15 dpc, and adult worms at 28 dpc [START_REF] Coadwell | Observations on the development of Haemonchus contortus in young sheep given a single infection[END_REF].

Parasitological examinations

Fecal samples from animals necropsied at 28 dpc were taken on days 21 and 28 post challenge to measure the nematode egg excretion. Fecal egg counts were performed according to the modified McMaster technique [START_REF] Raynaud | Étude de l'efficacité d'une technique de coproscopie quantitative pour le diagnostic de routine et le contrôle des infestations parasitaires des bovins, ovins, équins et porcins[END_REF]. After necropsies, the contents and washings of the abomasum were collected and passed through a 40 µm sieve to eliminate coarse materials. The whole abomasum was digested in pepsin-hydrochloric acid solution (37 °C, 6 h) to collect the tissue dwelling nematode stages. The contents, washings and digested materials, were preserved in absolute alcohol. The volumes of these materials were then adjusted to 1 L and worms were counted in a 10% aliquot and classified as adult male or female, immature male or female, or early and late L 4 . Twenty adult female worms were randomly picked from each 28 dpc sample for total length measurement and egg counts in utero. For this purpose, individual female worms were allowed to disintegrate in sodium hypochloride 4% solution [START_REF] Kloosterman | Counting eggs in utero of individual female worms[END_REF] and all eggs liberated from the uterus were counted for each worm.

Cytokine expression in abomasal

lymph node and fundic mucosa

mRNA extraction and RT-PCR

About 300 mg of the abomasal lymph node or fundic mucosa were snap frozen in (Invitrogen) and chilled on ice; a mixture containing M-MLV transcriptase (400 IU/ sample), 5× first-strand buffer (Life Technologies, Cergy-Pontoise, France), dithiothreitol (DTT) 0.1 M (Life Technologies), 40 U of RNase Out Ribonuclease Inhibitor (Invitrogen), 10 mM of each four dNTP (Promega) was then added and the mixture was incubated for 1 h at 42 °C. The reaction was heat-inactivated at 95 °C for 5 min and kept at -20 °C until use. For quantitative PCR, cDNA were used at dilutions of 1:100.

PCR primers

Specific primers and amplicon sizes are listed in Table II. For each target cDNA, a primer pair was determined using Primer Express Software (Applied Biosystems, Courtaboeuf, France) for SYBR Green realtime PCR assay on a GeneAmp 5700 Sequence Detection System (Applied Biosystems) and based on known ovine gene sequences (β-actin, IFN-γ, TNF-α, IL-3, IL-4, IL-5, IL-10, IL-12p40). Oligonucleotides were designed to amplify a product with a size of 51 bp, with a melting temperature (Tm) of 58-60 °C. When the ovine Ovine IL-3 AAGAATATCCTGGCGAATAAGAGC GAACGCTTTCAGGTTTGCCT gene sequence was not known (Eotaxin, IL-13), a consensus sequence was created, based on a minimum of three known sequences in other mammalian species. A first primer pair was then designed using Primer 3 Software in order to amplify the largest possible mRNA in all aligned sequences. PCR amplification on reverse transcript RNA obtained from ovine peripheral blood mononuclear cells (PBMC) stimulated with concanavalin A (ConA, 10 µg/mL, 24 h in a 5% CO 2 and 37 °C atmosphere) was then performed. Amplicons were purified before cloning in the TopoCloning system (Invitrogen) and sequenced (using M13 universal primers). The sequences thus obtained were then compared with the GenBank database using blast to validate the identity of the amplified product. After validation, a new set of primers suitable for quantitative PCR was designed using the obtained sequence. For IL-13, the primers used were those published by Hein et al. [START_REF] Hein | Long-term collection and characterization of afferent lymph from the ovine small intestine[END_REF].

Quantitative PCR

Quantitative PCR was performed on a GeneAmp 5700 (Applied Biosystems), using the double-stranded DNA binding dye SYBR Green I. PCR products corresponding to the different amplicons (see Tab. I) of target cytokines were cloned in PBR 2.1 plasmid (TopoClonA, Invitrogen) and sequenced (M13 universal primers). The plasmids harboring the cloned sequence were then quantified by spectrometry to establish the number of target copy/µL. Ten-fold dilution series were prepared from the stock plasmid solution corresponding to 10 7 to 10 1 copies of the target sequence. Serial dilution of the plasmid was then used as an external standard, allowing the determination of the number of copies of the target cDNA in each assay. Each sample for each investigated cytokine was performed in duplicate. Ovine Beta-actin was used as a housekeeping gene to normalize expression between different samples. The results obtained for each cytokine are expressed as a ratio of the number of cytokine mRNA copies/Beta-actin mRNA copies. A non-template control reaction (NTC) was included in each run. Finally, the specificity of the amplification reaction was assessed by acquisition of a dissociation curve. Data was obtained by slowly increasing the temperature of the PCR reaction from 60 to 95 °C; denaturation of nonspecific PCR products is followed by a faster decrease of fluorescence than for specific products. The melting profile of the distinct PCR product was obtained by plotting the first derivative (-dF/dT) of fluorescence (F) versus temperature (T).

Detection of antibodies in mucus and serum by indirect ELISA

A blood sample from each animal was recovered just before euthanasia and centrifuged for 5 min at 5 000 rpm. Serum was then frozen at -20 °C until analysis. A mucus sample from each animal was collected during necropsy using PBS impregnated filter paper strips of 4 cm 2 each deposited between the folds of the abomasal fundic mucosa for 5 min. The strips were then gently agitated in 2 mL of PBS for 2 h at room temperature to dilute the mucus. The liquid was centrifuged and stored frozen at -70 °C until analysis.

Worm antigen preparation

A donor sheep infected with 50 000 L 3 of H. contortus (Humeau strain) was sacrificed and adult worms were harvested from the abomasum. These worms were thoroughly washed in PBS (pH 7.4) containing penicillin (100 IU/mL) and streptomycin (1 mg/mL). Fifty adult worms per milliliter of the same buffer were maintained in a 5% CO 2 atmosphere at 37 °C overnight. Next, the supernatant (containing excretorysecretory products) was collected, filtered (0.2 µm) and stored at -70 °C until further use. A crude extract of H. contortus L 3 (CEL 3 ) was prepared after three cycles of freezing and thawing (-70 °C -+25 °C), homogenization at 4 °C and centrifugation at 30 000 g for 30 min at 4 °C. The supernatant was used as the crude extract of the L 3 antigen. The protein concentration of both antigenic preparations was determined with the method of Lowry et al. [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF].

Determination of optimal assay conditions and the ELISA technique

The optimal concentrations of ELISA reagents and the optimal test conditions were determined by chequerboard titrations of H. contortus antigens, positive (group A at 28 dpc) and negative (group C) reference sera or mucus and specific conjugates (Tab. III). An indirect ELISA was used on serum and mucus samples, as described by Jacquiet et al. [START_REF] Jacquiet | Regulation of Oestrus ovis (Diptera: Oestridae) populations in previously exposed and naïve sheep[END_REF]. The results are expressed as optical densities (OD) for serum and mucus IgG and IgA minus the OD of PBS wells (corrected absorbance).

Histology

At necropsy, two tissue samples from the fundic area of the abomasal wall were taken; one was fixed in 10% formalin for conventional histology and immunohisto-chemistry (IHC), while the second was stored at -70 °C for frozen IHC.

Conventional histology and IHC with paraffin-embedded samples

Abomasal tissue was paraffin-embedded and 3 µm sections were mounted on glass slides. Eosinophils and globule leukocytes were counted on haematoxylin-eosin stained slides whereas mast cells were counted after Giemsa staining. Immunohistochemistry was performed using antibodies already described for cell phenotyping on paraffin-embedded tissues in sheep [START_REF] Andreoletti | Phenotyping of protein-prion (PrPsc)-accumulating cells in lymphoid and neural tissues of naturally scrapie-affected sheep by double-labeling immunohistochemistry[END_REF][START_REF] Ramos-Vara | Reactivity of polyclonal human CD3 antiserum in lymphoid tissues of cattle, sheep, goats, rats and mice[END_REF][START_REF] Valentine | B-cell leukemia in a sheep[END_REF]. This antibody set allows specific labeling of macrophages/monocytes (mouse anti-human CD68 antibody, clone Ki-M6, Serotec, Cergy-Saint-Christophe, France, 1:150 dilution), B lymphocytes (mouse anti-human CD20-like antibody, clone BLA-36, Novocastra, Le-Perray-en-Yvelines, France, 1:50 dilution), or T lymphocytes (rabbit anti-human CD3 antibody, A 0542, DAKO, 1:100 dilution).

IHC on frozen samples

Five µm sections of abomasal tissue were obtained using a Leika cryomicrotome. Slides were fixed for 20 min in a For each type, cells were counted on five randomly selected fields at high magnification (× 400). The results are expressed as the sum of the five fields.

Statistical analysis

Comparisons between the three groups (previously-infected, primary-infected and uninfected control) and between the killing times (3, 7, 15 and 28 dpc) within a group were performed using the Kruskal-Wallis and Mann-Whitney non parametric tests (SYSTAT software). P < 0.05 was considered significant. For the 81 observations in this study, Spearman correlation coefficients (r) were statistically significant when r > 0.217 (P < 0.05) and r > 0.283 (P < 0.01).

RESULTS

Efficiency of experimental H. contortus challenge

No worm or egg excretions were observed in uninfected control animals throughout the study (group C). All lambs in groups A and B became infected after challenge with establishment rates ranging from 4% to 28%. Highly variable individual worm counts were observed within each group and there were no significant differences between groups A and B (Tab. IV).

Cytokine mRNA transcription in abomasal lymph node and in fundic mucosa

A clear increase of IL-4 m-RNA transcription was observed in the abomasal lymph node and fundic mucosa of both challenged groups but not in uninfected control sheep (Figs. 1A and1B). In the previously-infected group, an over-expression of IL-4 m-RNA was present as early as 3 dpc and remained significant and stable until the end of the experiment. In the primary-infected group, the IL-4 mRNA increase was not observed before 7 dpc but reached similar levels to previouslyinfected animals at 15 dpc. In fundic mucosa, IL-5 mRNA was higher (P < 0.05) in challenged lambs (groups A and B) than in uninfected control sheep at 7 dpc and 15 dpc (Fig. 1D). In the abomasal lymph node, the increase of IL-5 m-RNA was observed at 28 dpc only (data not shown). In fundic mucosa, IL-13 mRNA was higher in both challenged groups (A and B) than in uninfected control sheep (Fig. 1C) as early as 7 dpc, while no differences were recorded between the three groups in abomasal lymph node (data not shown). In both investigated tissues, no modifications of IL-3 or Eotaxin mRNA transcription were observed (data not shown). No significant group differences were recorded in the IFN-γ (Fig. 1E), IL-12 (Fig. 1F), IL-10 or TNF-α mRNA transcription profile during the whole experiment (data not shown). No cytokine mRNA transcription profiles could be directly correlated to the worm establishment rate, worm development or female fecundity.

Specific antibody detection in abomasal mucus and serum

At 7 and 15 dpc, a significant (P < 0.05) and specific CEL 3 IgA response (Fig. 2E) was observed in the mucus of previouslyinfected animals only. At 28 dpc, levels of specific CEL 3 IgA in mucus were similar in both infected groups and significantly higher than in uninfected controls (P = 0.01). In challenged groups, significant (P < 0.05) systemic IgG and IgA and local antibody responses against excretory-secretory products (ESP) of adult worms were observed at [START_REF] Gill | Induction of T helper 1-and T helper 2type immune responses during Haemonchus contortus infection in sheep[END_REF]

Dynamics of the cellular abomasal infiltration

In the abomasal mucosa of previouslyinfected sheep, eosinophils, mast cells and globule leucocytes were already present in significantly higher numbers than in primary-infected or uninfected control sheep (P < 0.01) at 3 or 7 dpc. In both challenged groups, a gradual increase of these cell populations was observed between 7 and 28 dpc. However, the cellular recruitment was delayed in the primary-infected group when compared to the previously-infected one. Eosinophil counts were higher in primaryinfected animals than those of uninfected control animals at 7 dpc, at 15 dpc for mast cells and at 28 dpc for globule leukocytes. At the end of the experiment (28 dpc), similar mucosal infiltrations (three cell types) were observed in previously-infected and primary-infected sheep (Figs. 3A, 3B and 3C). Significant correlations were established between mucosal IL-4 mRNA transcription level and recruitment of (i) eosinophils, mast cells and globule leucocytes at 3 and 7 dpc (P < 0.01), and (ii) mast cells and globule leucocytes at 15 dpc (P < 0.05). These correlations strongly suggest a relation between the Th 2 -cytokine level response and intensity and the rapidity of mucosal cellular infiltration. Concerning CD3+-T cells, BLA 36+-B cells, and CD68+-monocyte/macrophage cells, no significant differences between group A, B and C animals were observed (data not shown). The CD4+/CD8+ ratio (approximately 3:1) remained similar among the three groups and stable during the course of the experiment (data not shown).

Impact of immune responses on parasite populations

Similar total worm burdens in previously-infected and primary-infected lambs

No significant difference in the total worm burden was observed between Figure 3. Cellular infiltration in the abomasal mucosa (fundus): mast cells (A), globule leukocytes (B) and eosinophils (C), in previouslyinfected, primary-infected and uninfected control sheep (means and SD). For each cell type, the evaluation of cell number was performed on five randomly selected fields at high magnification (× 400). The results are expressed as the sum of five fields. a, b, c Values with no letter in common are significantly different (P < 0.05).

previously-infected and primary-infected sheep. Total worm burdens were not significantly correlated with antibody responses, or with eosinophils, mast cells or globule leukocytes (Tab. V).

Delayed worm development

At 3 and 7 dpc, only fourth-stage larvae (L 4 ) were observed in the challenged animals. Both early and late stage L 4 were observed at 7 dpc in groups A and B but the relative proportion of late L 4 was significantly (P < 0.01) increased in group B (78%) compared to group A (29%). Similarly, the mean ratio of immature worms/L 4 was 0.9 in primary-infected sheep and only 0.25 in previously-infected sheep (P < 0.01) at 15 dpc and several lambs from the primary-infected group already harbored adult worms while none were observed in the previously-infected group. At 28 dpc, the percentage of adult worms was higher in the primary-infected animals than in the previously-infected animals (Tab. IV). Taken together, these results suggest a delayed development of the parasite in previouslyinfected animals. The retarded development of early L 4 to late L 4 was associated with high eosinophil numbers in mucosa (7 dpc, r = 0.661, P < 0.01) and with increased IgA in the mucosa, both CEL 3 (r = 0.553, P < 0.01, at 7 dpc and r = 0.881, P < 0.01 at 15 dpc) and ESP specific (r = 0.712, P < 0.01 at 15 dpc). In the late phase of the experiment (28 dpc), eosinophils and mast cell populations were positively correlated with the proportion of immature worms. Eosinophil counts were also negatively correlated with the proportion of adult worms (Tab. V). Systemic ESP specific IgG response and mucosal CEL 3 specific IgA response were correlated with the proportion of immature worms (respectively r = 0.42, P < 0.05 and r = 0.59, P < 0.01).

Reduced female worm length and number of eggs in utero

In previously-infected sheep, adult females were shorter than in the primary-infected group (15.9 ± 2.1 mm versus 17.7 ± 2.7 mm) and contained fewer eggs in their uterus (174 ± 160.6 versus 275.4 ± 154.3 eggs per female) but the differences were not significant. Worm length and number of eggs in the uterus were highly correlated (r = 0.847). Increased eosinophils, mast cells and globule leukocyte populations were negatively correlated with the female worm length and the in utero egg numbers (P < 0.01) (Tab. V). Mucus CEL 3 specific IgA and systemic ESP specific IgG levels were correlated with the reduction of female worm length (respectively r = -0.36, P < 0.05 and r = -0.59, P < 0.01) (Tab. III). At 28 dpc, the number of egg-excreting animals was the following: 8 of group A (90 to 9 650 epg) and 9 of group B (300 to 25 000 epg). Despite the variability between sheep, egg excretions were significantly different between the two groups (P < 0.05) with a five-fold reduction in group A compared to group B. Eosinophils, mast cells and globule leukocyte numbers as well as the systemic IgG response against ESP of adult worms (r = -0.47, P < 0.05) were negatively correlated with the intensity of egg excretion in individual sheep (Tab. V).

DISCUSSION

The main objectives of this work were (i) to establish the polarization of the adaptive immune response in H. contortus infected sheep, (ii) to describe the kinetics of immune effector mechanisms following a single H. contortus infection in primaryinfected or previously-infected sheep and (iii) to study the effects of these responses on parasite establishment, development and females fecundity.

Few data were available on the orientation of the immune response in H. contortus challenged sheep. In a preliminary study using semi quantitative PCR, Schallig [START_REF] Schallig | Immunological responses of sheep to Haemonchus contortus[END_REF] observed a non-protective Th 1 response in primary-infected H. contortus infected sheep. A second challenge induced a protective Th 2 response with a high level of IL-4 mRNA expression without significant IL-5 over-expression. However, these results were obtained with a limited number of sheep. In our experiment, neither primaryinfected nor previously-infected H. contortus infected sheep showed a Th 1 cytokine pattern. In contrast, a clear over transcription of the IL-4 gene was observed in the abomasal lymph node and in the mucosa in both primary-infected and previouslyinfected lambs. Furthermore, specific IgG and IgA antibody responses in mucus and serum and higher mucosal recruitment of eosinophils, mast cells and globule leukocytes were observed in these two groups. Taken together, IL-4 mRNA over-transcription and immunoglobulin production/ cell recruitment strongly support a Th 2 orientation of the immune response in lambs challenged with H. contortus [START_REF] Balic | The immunobiology of gastrointestinal nematode infections in ruminants[END_REF]. In the present study, the over-expression of IL-5 and IL-13 in infected animals also suggested the Th 2 polarization. Recently, Pernthaner et al. [START_REF] Pernthaner | Increased expression of interleukin-5 (IL-5), IL-13, and tumor necrosis factor alpha genes in intestinal lymph cells of sheep selected for enhanced resistance to nematodes during infection with Trichostrongylus colubriformis[END_REF] described an increased expression of Th 2 related cytokines, IL-5 and IL-13 (but not IL-4), in intestinal lymph cells of sheep selected for enhanced resistance to T. colubriformis. Data collected in these two nematode models support the hypothesis of a Th 2 orientation in sheep, as reported in rodent [START_REF] Bancroft | Th1 and Th2 cells and immunity to intestinal helminths[END_REF][START_REF] Finkelman | Cytokine regulation of host defense against parasitic gastrointestinal nematodes: lessons from studies with rodent models[END_REF] or in human [START_REF] Cooper | Human infection with Ascaris lumbricoides is associated with a polarized cytokine response[END_REF] gastro-intestinal nematode infections.

The second objective of our study was to investigate the dynamics of the adaptive immune response after a single H. contortus infection. Repetitive or continuous infections are usually used to study the host immune response and to mimic natural infections as well as possible [START_REF] Adams | Infection with Haemonchus contortus in sheep and the role of adaptive immunity in selection of the parasite[END_REF][START_REF] Balic | Mechanisms of immunity to Haemonchus contortus infection in sheep[END_REF][START_REF] Barger | Regulation of Haemonchus contortus Th 2 response in H. contortus infected sheep 621 populations in sheep exposed to continuous infection[END_REF]. Nevertheless, single H. contortus infections were necessary in our experiment which was mainly designed to measure host immune response effects on worm development. IL-4 mRNA expression increased gradually from 7 dpc to 28 dpc in primaryinfected sheep but remained stable and high in previously-infected sheep from 3 to 28 dpc. Eosinophil, mast cell, and globule leukocyte recruitments were apparent at 7, 15 and 28 dpc respectively in primaryinfected animals while these cells were still present in previously-infected lambs at 3 dpc. Antibody responses were delayed and low in primary-infected lambs and rapid and higher in previously-infected ones as previously shown by Schallig et al. [START_REF] Schallig | Isotype-specific serum antibody responses of sheep to Haemonchus contortus antigens[END_REF] and Gomez-Munoz et al. [START_REF] Gomez-Munoz | Serum antibody response of Castellana sheep to Haemonchus contortus infection and challenge: relationship to abomasal worm burdens[END_REF]. Consequently, while final response intensities were similar in both infected groups, recruitment of cells and secretion of immunoglobulins in previously-infected sheep were observed before their occurrence in primary-infected sheep. This difference could be explained by (i) a rapid mobilization of immune mechanisms after challenge in group A compared to group B or from (ii) the presence of remaining cells and antibodies induced by the first infection. Unfortunately, our experimental design did not allow to state between these two hypotheses. Interestingly, this earlier mobilization of the immune effectors was associated with significant higher IL-4 mRNA levels at 3 dpc in previously-infected sheep.

No differences in the establishment rates of worms were observed between previously-infected and primary-infected lambs in this experiment. Our data provide evidence that the parasite development and the female fecundity were depressed in previously-infected animals compared to primary-infected ones. This delay in parasite development has been shown after repeated exposure of lambs to H. contortus [START_REF] Schallig | Isotype-specific serum antibody responses of sheep to Haemonchus contortus antigens[END_REF] or T. circumcincta [START_REF] Stear | Regulation of egg production, worm burden, worm length and worm fecundity by host responses in sheep infected with Ostertagia circumcincta[END_REF]. Here we established that alterations of parasite life traits (development of worm and female fecundity) are related to the earliness of effector mobilization rather than to the final level of the responses. Eosinophils, mast cells and globule leucocyte recruitment are typically associated with helminth infections [START_REF] Balic | Cellular profiles in the abomasal mucosa and lymph node during primary infection with Haemonchus contortus in sheep[END_REF]. In our study, these cells were strongly and negatively correlated with adult female length and number of eggs in utero. Moreover, positive correlations were also observed between mast cell and eosinophil numbers and the relative proportion of immature worms while a strong negative correlation was seen between eosinophils and the proportion of adult worms (Tab. V). Thus, these cells may have a role in the delayed development of the parasite and the decrease of fecal egg output as previously shown by many authors [START_REF] Balic | The immunobiology of gastrointestinal nematode infections in ruminants[END_REF][START_REF] Dawkins | Eosinophil responses in sheep selected for high and low responsiveness to Trichostrongylus colubriformis[END_REF][START_REF] Terefe | Haemonchus contortus egg excretion and female length reduction in sheep previously infected with Oestrus ovis (Diptera: Oestridae) larvae[END_REF]. In the present study, two antibody responses (CEL 3 specific IgA response in abomasal mucus and ESP specific IgG in serum) were positively correlated to the proportion of immature worms and negatively correlated to the female length and number of eggs in utero suggesting that they could interfere with H. contortus development within the host. The importance of the local IgA response in host protection has already been indicated in T. circumcincta infected sheep, where the production of local IgA against somatic or L 4 ESP molecules induced a reduction in the female parasite length and fecundity [START_REF] Stear | Regulation of egg production, worm burden, worm length and worm fecundity by host responses in sheep infected with Ostertagia circumcincta[END_REF].

  previously-infected and challenged sheep; Group B: primary-infected sheep. Six animals per group were sacrificed at 3, 7 and 15 days post challenge (dpc), and 11 and 10 sheep at 28 dpc for groups A and B respectively. Coprological examinations were performed at 21 and 28 dpc for sheep sacrificed at 28 dpc.

Figure 1 .

 1 Figure 1. Th 1 /Th 2 cytokine mRNA transcription in the abomasal lymph node (ALN) and in the fundic mucosa (AFM) from previously-infected, primary-infected and uninfected control sheep. IL-4 mRNA (A: in ALN and B: in AFM), IL-13 mRNA in AFM (C), IL-5 mRNA in AFM (D), IFN-γ in ALN (E) and IL-12 in ALN (F) were quantified and measured in each duplicate sample using an external standard. The results are expressed after normalisation (ratio) with a housekeeping gene (ovine Beta-actin) (means and SD). a, b, c Values with no letter in common are significantly different (P < 0.05).

Figure 2 .

 2 Figure 2. Local and systemic IgG and IgA responses to parasite (excretory-secretory products (ESP) of H. contortus adult worms or crude extract of L 3 (CEL 3 )) antigens, in previously-infected, primaryinfected and uninfected control sheep. A: Total mucus IgG against ESP; B: Total systemic IgG against ESP, C: Mucus IgA against ESP; D: Systemic IgA against ESP, E: Mucus IgA against CEL 3, F: Systemic IgA against CEL 3 (means and SD). a, b, c Values with no letter in common are significantly different (P < 0.05).

Table I .

 I Schematic description of the experimental design.

	Treatment and group	Initial infection*	Levamisole Challenge infection*	Number of sheep killed
	Days from challenge	-35		-7	0	3	7	15	28
	Previously-infected (A)	+		+	+	6	6	6	11
	Primary-infected (B)	-		+	+	6	6	6	10
	Uninfected control (C)	-		+	-	6	6	6	6
	*10 000 H. contortus L 3 .
	1 mL of Trizol Reagent (Invitrogen, Cergy-
	Pontoise, France, reference 15596-018),
	and stored at -70 °C before use. Samples
	were homogenized using a Hybaid RiboLy-
	ser TM (two cycles of 30 s each at a speed of
	4.5). Fifty µL of the obtained homogenate
	were mixed with 950 µL of Trizol before
	adding 200 µL of chloroform at room tem-
	perature for 10 min. The samples were then
	centrifuged for 15 min at 20 000 g at 4 °C.
	The supernatants were placed on an RNe-
	asy Column (RNeasy Mini Kit, Qiagen,
	Courtaboeuf, France) and then processed
	using the Qiagen protocol for RNA clean-
	up. This complete protocol allows the
	recovery of highly pure RNA. The RNA
	concentration was measured by spectro-
	photometry at 260 nm and RNA quality was
	assessed by electrophoresis on 1.2% agar-
	ose gels stained with ethidium bromide.
	Two µg of total RNA were digested with 1U
	of RNase-free DNAse (Promega, Charbon-
	nières, France) for 1 h at 37 °C to remove
	any trace of genomic DNA, and then incu-
	bated 10 min at 65 °C with DNase Stop
	Solution. Treated RNA was used as the
	template for single-stranded cDNA synthe-
	sis. They were incubated 10 min at 65	°C
	with Random Primer oligonucleotides

Table II .

 II Forward and reverse primer sequences for quantitative reverse-transcriptase (RT) PCR.

	Cytokine	Forward sequence	Reverse sequence	Tm amplicon (°C)
	Ovine Beta-actin	ACCAGTTCGCCATGGATGAT	AGCCGTTGTCAACCACGAG	78
	Ovine IFN-γ	ATCTCTTTCGAGGCCGGAGA	ATTGCAGGCAGGAGAACCAT	77
	Ovine IL-4	GGAGCTGCCTGTAGCAGACG	TTCTCAGTTGCGTTCTTTGGG	79
	Ovine IL-10	CTGAGAACCATGGGCCTGAC	TCTCCCCCAGCGAGTTCAC	80
	Ovine TNF-α	CCCGTCTGGACTTGGATCCT	TGCTTTTGGTGCTCATGGTG	75
	Ovine IL-12p40	GAATTCTCGGCAGGTGGAAG	GTGCTCCACGTGTCAGGGTA	80
	Ovine IL-13	AGAACCAGAAGGTGCCGCT	GGTTGAGGCTCCACACCATG	80
	Ovine Eotaxin ACAAGAAAATCTGTGTTGATCCCC	CCATGGCATTCTGGACCC	75
	Ovine IL-5	CTGCTGATAGGTGATGGGAACTT GGTGATTTGTATGCTGAGGAGTAGG	74

Table III .

 III Reagents and procedures used for antigen ELISA detection in serum and mucus.Type of antigens: ESP Ad: Excretory-secretory products from H. contortus adult worms; CEL 3 : crude extract of H. contortus infective third-stage larvae. For IgG determination, the secondary antibody is a Donkey anti-sheep IgG HRP-conjugated. For IgA determination, an anti-IgA bovine / sheep mouse IgG 1 is first applied before a goat anti-mouse IgG1 HRP-conjugated.

		Th 2 response in H. contortus infected sheep			613
	-20 °C acetone bath before drying. Slides
	were then directly processed for IHC. Mono-
	Serum clonal antibodies directed against CD4			Mucus
	Isotype (mouse anti-ovine CD4, SEROTEC, refer-IgG ence MCA2213, 1:2000 dilution), or CD8	IgA	IgG		IgA
	Type of antigen (mouse anti-bovine CD8, SEROTEC, ref-ESP Ad CEL3 ESP Ad CEL3	ESP Ad CEL3	ESP Ad CEL3
	erence MCA837G, 1:800 dilution) antigens Concentration of antigen (µg/mL) 2 2 5 were used.	2	5	0 . 5	5	2
	Dilution of serum or mucus	1:100 1:100	1:5	1:5	1:1	1:1	1:1	1:1
	Dilution of secondary antibody	1:1000 1:1000	1:100 1:100	1:1000 1:1000	1:250 1:250
	Dilution of conjugate	-	-	1:500 1:500	-	-	1:500 1:500

Table IV .

 IV and 28 dpc (Figs. 2A, 2B, 2C and 2D). No Egg excretion, total worm burden, length and number of eggs in utero of adult female worms, stage and sex composition of Haemonchus con-

		Adult	females	(%)
		Adult	males	(%)
		Immature	females	(%)
		Immature	males	(%)
		Late	L4	(%)
		Early	L4	(%)
		Number of	eggs in utero	(mean ± SD)
		Adult female	length (mm)	(mean ± SD)
		Total	worm burden	(mean ± SD)
		Epg	at 28 dpc	(mean ± SD)
		Epg	at 21 dpc	(mean ± SD)
	tortus populations.	Days post Group challenge

Table V .

 V Correlations between abomasal eosinophils, mast cells or globule leukocytes and worm parameters 28 days post challenge.

	Th 2 response in H. contortus infected sheep	619
	3.5.4. Reduced fecal egg output
		Eosinophils	Mast cells	Globule leukocytes
	% L 4 worms	0.308	0.189	0.019
	% Immature worms	0.431*	0.439*	0.297
	% Adults worms	-0.413*	-0.332	-0.170
	Total worm burden	-0.086	-0.102	-0.129
	Adult female length	-0.589**	-0.508**	-0.676**
	Number of eggs in utero of adult females	-0.477**	-0.486**	-0.592**
	Fecal egg output	-0.378*	-0.402*	-0.369*
	* P < 0.05 and ** P < 0.01.

ACKNOWLEDGEMENTS

We are very grateful to Gilles Aumont, Getachew Terefe and Mike Stear for their valuable contributions to the present manuscript. We are very indebted to the technical staff of La Sapinière INRA experimental station (INRA, Department of Animal Genetics) for rearing and providing INRA 401 lambs. This work was supported by the FEOGA program (Région Centre) "Résistance génétique aux nématodes".