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Physiol. Genomics 30: 253-261, 2007. First published May 8, 2007; doi:10.1152/physiolgenomics.00273.2006 Free Article
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Received 11 December 2006; accepted in final form 8 May 2007.
Physiological Genomics 30:253-261 (2007)
1094-8341/06 $8.00 © 2007 American Physiological Society

Transcriptional profiling of Ovis aries identifies Ovar-DQA1 allele frequency differences between nematode-resistant and susceptible selection lines

Orla M. Keane1, Ken G. Dodds2, Allan M. Crawford2 and John C. McEwan2

1 AgResearch Molecular Biology Unit, Department of Biochemistry, University of Otago, Dunedin
2 AgResearch Invermay Agricultural Centre, Mosgiel, New Zealand


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Gastrointestinal nematodes are a major cause of disease in grazing livestock; however, individual animals differ in their response to infection. To identify genes whose expression correlates with resistance status, transcriptional profiling of resistant and susceptible sheep was undertaken. Transcription profiles were taken at three time points during the growth of lambs. The number of genes differentially expressed increased as animals were exposed to longer nematode challenge. Almost 300 genes, with a variety of functions, were differentially expressed overall, although genes more highly expressed in resistant animals typically had major histocompatibility complex (MHC) II, free radical scavenging or smooth muscle-specific functions. The Ovar-DQA1 gene was 8.4-fold more highly expressed in resistant animals. This was due in part to a higher frequency of DQA1 null alleles in susceptible animals. The null allele of DQA1 was also associated with susceptibility in a separate selection flock, presenting the hypothesis that failure to present parasite antigens to immune cells led to nematode susceptibility. To test this hypothesis, commercial rams from three breeds were genotyped for the null allele of DQA1. The homozygous null allele was associated with susceptibility in only one of the three breeds tested indicating that the null allele does not cause susceptibility to intestinal parasites per se but is probably in linkage disequilibrium with additional polymorphisms in the MHC region. A combination of these polymorphisms may contribute to susceptibility in some populations. The extent of linkage disequilibrium between polymorphisms may vary from breed to breed or population to population.

sheep; gastrointestinal parasite; major histocompatibility complex; microarray


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GASTROINTESTINAL NEMATODES are one of the most serious causes of disease in grazing ruminants and are of concern in both developed and developing countries. The economically important parasites belong to the order Strongylida and family Trichostrongyloidea and include Haemonchus, Teladorsagia (Ostertagia), Nematodirus, Cooperia, and Trichostrongylus species. Infection with these nematodes leads to clinical disease and lost production. Prophylactic measures, such as anthelmintic drenching, have historically been used to control nematode infection. However, the efficacy of this treatment is decreasing as anthelmintic resistant nematodes evolve. Indeed, nematodes resistant to all major classes of anthelmintics have now been isolated (28). The presence of chemical residues in food products is also increasingly undesirable (30). Novel forms of nematode control are urgently required.

As individual animals or breeds are known to differ in their resistance to gastrointestinal nematodes and resistance has been shown to be a heritable trait (37), a natural method of parasite control is to select for resistant animals. Although resistance is primarily an immune based Th2-type response (10, 32, 41, 50), resistance to one species of gastrointestinal nematode often confers resistance to the other major species (22, 48, 55), primarily through a nonspecific mechanism of parasite expulsion (7). Selective breeding for host resistance has made significant genetic gains in a number of sheep breeds (9, 33, 37, 53, 56). Breeding is, however, currently based on the phenotype of the fecal egg count (FEC), which is costly and cumbersome to measure and has only a moderate heritability (h2 = 0.23–0.41) (18). Genetic gain could be accelerated if the gene variants responsible for resistance were identified and animals were selected on the basis of genotype. A number of linkage studies have been carried out to map genes that confer resistance to gastrointestinal nematodes (8, 14, 15). Association studies have also shown the major histocompatibility complex (MHC), interferon-{gamma}, and immunoglobulin E regions as loci associated with resistance to internal nematodes (11–13, 39, 44–46).

The MHC encoded on sheep chromosome 20 plays a pivotal role in the host response to infection. MHC encodes cell surface-expressed glycoproteins, which present antigens to immune cells. MHC I, present on the majority of cell types, presents endogenously derived peptides to CD8+ T cells. MHC II, in contrast, is expressed on the surface of antigen presenting cells and presents exogenously derived peptides to CD4+ T cells. The MHC region is highly polymorphic and under balancing selection (38, 42). Markers in both MHC I and MHC II regions have shown association with FEC (11, 49). A number of MHC II genes have also been shown to be more highly expressed in the duodenum of parasite-resistant animals compared with susceptible (16).

Transcriptional profiling has been used successfully to identify genes responsible for particular phenotypes (2). In this study we have examined relative gene expression levels in lines of Perendale sheep divergently selected for resistance or susceptibility to gastrointestinal nematodes. This was done using high-throughput DNA microarray technology. This enabled us to identify genes and pathways that are differentially expressed between resistant and susceptible animals and so may contribute to an animal's phenotype.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Selection lines and tissue collection.
Resistant and susceptible lines of Perendale sheep have been selected at AgResearch, New Zealand since 1986. These lines now differ in FEC by 4.9-fold (37). Resistant and susceptible lambs born in each of the years 2000–2002 were cograzed to allow natural exposure to gastrointestinal parasites at an AgResearch farm at Woodlands, New Zealand. At an average age of 276 days for animals born in 2000, 175 days for animals born in 2001, and 84 days for animals born in 2002, four lambs per selection line were chosen and these animals were killed. A 5-mm cross section of duodenum tissue, 20 cm distal to the pyloric sphincter, was collected. The tissue was frozen in liquid nitrogen and stored at –80°C. For each animal its fecal egg output was recorded as were the number of adult nematodes in the abomasum and duodenum at the time of euthanasia (Table 1). All procedures were approved by the AgResearch Invermay Animal Ethics Committee.


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  		Table 1. Adult worm burden and FEC for animals used in the expression array study

 
RNA preparation.
Total RNA was isolated from the duodenum of each animal with TRIzol (Invitrogen) and was cleaned using an RNeasy kit (Qiagen). Labeled cDNA was made from 20–35 µg of total RNA using the SuperScript Indirect cDNA Labeling System (Invitrogen) and Cy3 and Cy5 mono-reactive dyes (Amersham) according to the manufacturer's instructions.

Array preparation, slide hybridization, scanning, and normalization.
Two ovine cDNA arrays, consisting of 11,520 and 19,968 spots, were prepared from ovine expressed sequence tag (EST) libraries as described previously (29). Gene expression between four resistant and four susceptible animals was compared at each time point (T = 84, 175, 276 days) by hybridizing fluorescently labeled cDNA to either the ovine 11k array (T = 84, 175 days) or the ovine 20k array (T = 276 days). The 11k array was a subset of the 20k array. Gene expression between resistant and susceptible lambs born in 2000 has been examined previously (16) albeit with a smaller bovine EST array. The experimental design at each time point was a factorial dye swap design involving 16 slides, where every animal was compared with every animal in the opposite selection line as described previously (16). Slides were prehybridized, hybridized, washed, and scanned as described previously (29). Data for each slide were normalized following the procedure of Baird et al. (6). The slides from the three time points were then combined into a meta-analysis, resulting in a 48-slide experiment using all 24 animals. ESTs were excluded from the analysis if information about that EST came from <12/48 spots. ESTs that lay outside the 95% confidence limit for the log ratio of the T value were counted as differentially expressed (Supplementary Table S1). 1 In total, 408 ESTs were more highly expressed in the duodenum of susceptible animals, while 81 ESTs were more highly expressed in the duodenum of resistant animals. The 489 differentially expressed ESTs were then re-PCR amplified from the original clone. We excluded 102 from subsequent analysis, as they did not give a single clear band on reamplification. All the microarray information has been submitted into the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus website. The accession number for the experiment series is GSE5492.

Quantitative RT-PCR.
First-strand cDNA synthesis, from 2 µg of total RNA, was carried out using Superscript III (Invitrogen) according to the manufacturer's instructions. The cDNA was diluted 1:12.5 before use. For each ovine transcript to be quantified, the putative intron-exon boundaries were determined from the human boundaries. Primers that amplified across these boundaries were designed using Primer Express 2.0 (Applied Biosystems). For each gene the primer pairs were titrated to determine optimal primer concentration (Supplementary Table S2). PCR was carried out in triplicate in 20-µl reactions containing 1x SYBR Green qPCR SuperMix (Invitrogen), 0.1x ROX, an appropriate concentration of primers, and 5 µl cDNA. Reactions were heated to 50°C for 2 min and then denatured to 95°C for 10 min. Reactions were then cycled 40 times at 95°C for 15 s and 60°C for 1 min using the 7500HT sequence detection system (Applied Biosystems). A dissociation analysis was carried out at the end of each reaction to ensure a single PCR product was generated. Relative mRNA levels were quantified by comparing to a standard curve generated using cDNA derived from an unrelated Perendale animal, and mRNA levels were normalized to that of PolR2E. Standard deviations for individual animals were calculated by the delta method (19), and selection line differences were tested with an unpaired one-sided t-test.

Microarray data interpretation.
All ESTs in the ovine libraries, along with all ovine ESTs deposited in NCBI, were assembled into contigs using CAP3 (26) after an initial clustering step using basic local alignment search tool (BLAST) (3). ESTs on the array were annotated by finding the human RefSeq (RefSeq release at 5/2005) corresponding to the contig to which they belonged using BLASTN and the following options: -e 0.0000000001 -v 5 -b 5. Each EST was annotated with the top human RefSeq hit. In cases where the EST matched more than one transcript variant of a gene then the top hit is listed. Approximately 85% of the ESTs on the array could be annotated in this manner. Of the 387 differentially expressed ESTs, 323 could be annotated. For those annotated genes with associated Gene Ontology (GO) terms, these terms were extracted and GO terms significantly associated with the differentially expressed genes were found using EASE (25).

Ovar-DQA1 amplification and sequencing.
A 470-bp region from intron 1 to intron 2 of DQA1 was amplified from genomic DNA using the primers DQA1exon2F and DQA1exon2R. Reactions were carried out in 1x PCR buffer with 2 mM MgCl2, 0.2 mM each dNTP, 0.4 µM of each primer, 2.5 U Taq polymerase (Invitrogen), and 100 ng genomic DNA. The reactions were denatured at 95°C for 2 min and then cycled 36 times at 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min. Reactions were then extended at 72°C for 10 min. A 300-bp region from exon 1 to exon 2 of DQA1 was amplified from cDNA using the primers DQA1seqF and DQA1seqR. Reactions were carried out as described above except using 3 mM MgCl2, an annealing temperature of 53°C, and 30 cycles. Templates for sequencing were cleaned using the High Pure PCR product clean up kit (Roche). Sequencing reactions were carried out using the BigDye terminator v3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions. Reactions were analyzed on an ABI3100 Genetic Analyzer.

Southern blotting.
Genomic DNA (5 µg) was digested at 37°C for 6 h with the restriction enzyme EcoRI (Roche). Fragments were separated by electrophoresis through a 1% agarose gel. After depurination, denaturation, and neutralization fragments were transferred to a nylon membrane (Hybond-N+) by capillary transfer. DNA was fixed to the membrane by UV cross-linking. A probe for Southern blotting was generated by amplification of ~265 bp from intron 1 to exon 2 of DQA1 using the primers DQAintronF2 and DQAexonR2 as described above except using 1 mM MgCl2, an annealing temperature of 50°C, and an extension time of 30 s. This probe was subsequently labeled with {alpha}-[32P]dCTP (Amersham) using the RadPrime DNA labeling system (Invitrogen) according to the manufacturer's instructions. Prehybridization, hybridization, and washing were carried out as described previously (4).

Haplotyping.
Ovar-DQA1 haplotypes were determined using 154 bp of DNA sequence commencing at position 1 of exon 2. When different sequences were obtained from genomic and cDNA, it was assumed that this was due to allele-specific amplification, and each sequence was taken as one of the haplotypes. Haplotypes for those animals that were heterozygous using both sequencing methods were inferred by assuming they were previously known haplotypes. Known haplotypes either were observed in this study as homozygotes or were from the published literature. Haplotypes assigned under this assumption were unambiguous. The frequency of the null allele was calculated assuming Hardy-Weinberg equilibrium. Phylogenetic trees were generated by the maximum parsimony method, and bootstrap support for branches was calculated using the phylogeny inference package PHYLIP.

Haplotype frequency differences.
Haplotype frequency differences between selection lines were tested with Peddrift, a program that calculates exact probabilities of allele frequency differences taking into account random genetic drift and founder sampling effects observed in selection lines (17). Haplotypes with low frequency (<10%) were combined with a neighboring group, using the estimated phylogeny, following the principles of Templeton et al. (51). This strategy was also followed to combine haplotypes to allow more powerful comparisons.

Commercial populations.
Whole blood was collected from 432 progeny-tested sires from commercial farms. These animals had estimated breeding values for FEC1 (fecal egg count at 4–5 mo of age) and FEC2 (fecal egg count at 6–8 mo of age). Typically each sire had 10–50 progeny, and breeding values used were obtained from a national across flock and breed genetic evaluation (Sheep Improvement Limited)(1). DNA was extracted from the blood as described previously (34), and animals were genotyped as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Using transcriptional profiling, we measured gene expression at three time points during the growth of Perendale sheep selected for resistance or susceptibility to gastrointestinal nematodes. It was found that no genes were significantly differentially expressed between the resistant and susceptible animals at weaning (T = 84 days). A small number of genes were differentially expressed when the animals were exposed to challenge (T = 175 days; 7 and 77 genes in resistant and susceptible animals respectively), while many more genes were differentially expressed as animals were exposed to challenge over a longer period (T = 276 days; 134 and 436 genes in resistant and susceptible animals, respectively). Data from all time points were combined, and genes that were significantly differentially expressed overall were detected. This analysis identified almost 300 genes that were differentially expressed across a number of ages and exposure levels, and so these genes represent a set of genes of general effect.

In total, 272 annotated ESTs were more highly expressed in susceptible animals (Supplementary Table S3) and 51 annotated ESTs were more highly expressed in resistant animals (Supplementary Table S4). In some cases two or more differentially expressed ESTs corresponded to the same human RefSeq gene. This resulted in 247 unique genes that were more highly expressed in susceptible animals and 50 unique genes that were more highly expressed in resistant animals. To independently validate the results of the microarray experiment, we chose 10 genes to confirm differential expression by real-time RT-PCR. In total, eight of these genes confirmed differential expression (P < 0.05) between resistant and susceptible animals (Table 2). One gene, catalase, showed increased expression although it did not reach significance (P = 0.07), and one gene was not confirmed to be differentially expressed.


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  		Table 2. Confirmation of microarray results for 10 genes by quantitative RT-PCR

 
GO terms associated with genes more highly expressed in resistant animals are listed in Table 3. The most significant of these relate to MHC class II activity, exogenous antigen processing, and presentation. Other GO terms relate to lipid metabolism and oxidoreductase activity. GO terms associated with genes more highly expressed in susceptible animals are listed in Table 4 and relate to the stress response such as heat shock protein activity, chaperone activity, and remodeling chromatin organization.


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  		Table 3. GO terms significantly associated with genes more highly expressed in resistant animals

 

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  		Table 4. GO terms significantly associated with genes more highly expressed in susceptible animals

 
It has previously been reported that the smooth muscle-expressed genes transgelin (TAGLN) and enteric {gamma}-actin (ACTG2) were more highly expressed in resistant animals than susceptible (16). We also found transgelin to be more highly expressed in resistant animals as well as the Wiskott-Aldrich syndrome family gene 2 (WASF2), myosin light chain kinase (MYLK), and desmin (DES). These genes are all expressed in the intestinal smooth muscle layer. We therefore compared the structure of the duodenum tissue in four resistant and four susceptible animals by hematoxylin and eosin staining. No gross morphological differences were seen in the mucosa or underlying muscle layer between the two lines (data not shown).

The MHC class II gene Ovar-DQA1 was expressed 8.4-fold more highly in resistant animals than susceptible animals (Table 2). In particular, eight susceptible animals and two resistant animals had extremely low or nondetectable levels of DQA1 expression (Fig. 1). This gene is known to be extremely polymorphic in many species, and 10–18% of sheep are reported to completely lack a DQA1 gene.(20, 47, 57). To determine if our animals were lacking DQA1 or simply not expressing it, a 470-bp region from intron 1 to intron 2 of DQA1 was amplified from genomic DNA from 52 animals from our selection lines. These animals comprised 26 resistant and 26 susceptible animals and included all 24 animals used in our expression array study. This genotyping method detects only null homozygous animals (NN) and cannot distinguish null heterozygotes from those with two copies of DQA1. This region of DQA1 could be amplified from 38 of the 52 animals (22 resistant and 16 susceptible; Fig. 2A), indicating that the remaining animals (four resistant and 10 susceptible) lacked a DQA1 gene.


Figure 1
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  		Fig. 1. Plot of relative DQA1 expression levels in resistant and susceptible animals. Quantitative PCR was used to measure DQA1 expression in duodenum tissue of the 12 resistant and 12 susceptible animals used in the microarray study. Error bars show the SD among replicate wells.

 

Figure 2
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  		Fig. 2. A: assay for homozygous DQA1 null animals. Exon 2 of DQA1 was amplified by PCR from 52 Perendale selection line animals. A band could not be amplified from genomic DNA of 14 of the animals indicating they lack a DQA1 gene. B: Southern blot confirming the absence of DQA1 from the genome of null animals. Genomic DNA from 9 animals was probed for DQA1. A hybridizing band of 3,800 bp was seen for 4 animals from which DQA1 could be amplified by PCR (DQA1 +), while this band was absent from the genome of 5 animals from which DQA1 could not be amplified (DQA1 –).

 
To determine if the animals that appeared null for DQA1 were actually missing the gene or if they were simply failing to amplify it due to mispriming of the oligonucleotides, a Southern blot was carried out. Due to the lack of sequence data for sheep and the polymorphic nature of the MHC II region, the size of the expected fragment was not known; however, four animals from which DQA1 could be amplified by PCR gave a band of ~3,800 bp. This band was not detected from five animals that failed to amplify DQA1 (Fig. 2B), indicating that DQA1 is indeed missing from the genome of these animals as described previously (23, 47, 57). Interestingly, DQA1 expression was still higher in resistant animals carrying a DQA1 gene than susceptible animals carrying a DQA1 gene (P = 0.002).

To determine which haplotypes were present in the Perendale selection line population, we sequenced part of exon 2 of DQA1 from the 38 animals carrying at least one copy of the gene. The PCR products from Fig. 2A were sequenced along with a PCR product from cDNA. We detected 22 polymorphisms in a 154-bp region commencing at position 1 of exon 2. Both cDNA and genomic DNA were scored for the polymorphisms. For seven of the animals, different haplotypes were detected from cDNA and genomic DNA. This indicated that allele-specific amplification had occurred with one haplotype preferentially amplified from cDNA and the other haplotype from genomic DNA. These animals were scored as heterozygous at the DQA1 locus. Two animals were detected as heterozygous in the cDNA sequence only, while a further two animals were detected as heterozygous in both cDNA and genomic DNA sequence; 27 animals appeared to be homozygous at DQA1. It was estimated, assuming Hardy-Weinberg equilibrium, that ~24 of these animals are in fact heterozygous carrying one copy of DQA1 and a null allele. However, the possibility remains that some of these animals carry a second allele of DQA1 that was not amplified from either genomic or cDNA due to allele specific amplification. Eight distinct DQA1 haplotypes were identified and two of these haplotypes have not been reported previously. An alignment of the haplotypes is shown in Fig. 3A, and the distance between the haplotypes is shown in Fig. 3B. The phylogeny shown in Fig. 3B is one of three that required a minimum of 60 base changes (31 within the ovine sequences) but is the one given as the consensus tree following bootstrapping. Each of the eight haplotypes identified gave rise to a unique amino acid sequence of the encoded protein.


Figure 3
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  		Fig. 3. A: Alignment of DQA1 haplotypes. Sequences are aligned between bases 1 and 154 of exon 2. A dot indicates identity with the top sequence. The nomenclature of the known haplotypes is based on that previously published (57), while newly identified haplotypes are named with their GenBank accession number. B: maximum parsimony tree of DQA1 haplotypes. The tree was constructed using the sequences displayed in A and 1,000 bootstrap replications. The numbers at the branch points indicate the bootstrap confidence values, and branch lengths are proportional to genetic distance. The human HLA-DQA1 RefSeq NM_002122 was used as an outgroup to root the tree.

 
The nominal DQA1 haplotype frequencies differed between resistant and susceptible animals, indicating that there was association between resistance to internal parasites and DQA1 haplotype (Table 5; P < 0.0001). However, Peddrift analysis (17) was found to be not significant at the 5% threshold (P = 0.065) even after grouping nonnull alleles to find the most significant contrast (combining all but DQA1*0103). However, testing within lines demonstrated that resistant animals lacking the DQA1 gene had higher FECs than resistant animals carrying a DQA1 allele (P = 0.0137). In addition, 100 animals from Romney selection lines that have also been selected for resistance or susceptibility to gastrointestinal nematodes and differ in FEC by 40-fold (36) were genotyped for the presence or absence of DQA1 using PCR as described above. In this case an association was found, with the null allele having a significantly higher frequency in the susceptible line (Table 5; P = 0.003) after accounting for genetic drift using Peddrift.


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  		Table 5. Frequencies of DQA1 haplotypes among the resistant and susceptible selection line animals

 
As the null allele was more frequent in susceptible animals in two independent selection lines this presented the attractive hypothesis that the absence of DQA1 led to susceptibility due to failure to present parasite antigens to CD4+ T cells. To test this hypothesis, 432 progeny-tested commercial sires from the Perendale, Romney, and Coopworth breeds were genotyped by PCR for the presence or absence of DQA1. In total, 335 of these animals carried at least one copy of DQA1, while the remaining 97 animals were homozygous for the null allele. Therefore, ~22% of the animals lacked a copy of DQA1. However, the homozygous null allele of DQA1 was significantly associated with susceptibility to intestinal nematodes in only one of the three breeds tested (Perendale; Table 6). Therefore it appears that the lack of DQA1 does not cause nematode susceptibility per se. Susceptibility to gastrointestinal nematodes is likely to be highly complex and may be due to another MHC allele linked to OLA-DQA1, a combination of susceptible MHC alleles, a non-MHC allele in linkage disequilibrium with DQA1, the level of expression of DQA1 on antigen presenting cells, or indeed a combination of all of these factors. Additionally the mechanism of resistance may differ in different genetic backgrounds.


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  		Table 6. Association of homozygous DQA1 null allele with breeding values for susceptibility to nematodes in commercial populations

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study identified almost 300 genes differentially expressed between parasite resistant and susceptible animals. As gene expression was examined at different time points in lambs born over 3 yr, the age of the animals was confounded with the environment. Additionally, more differentially expressed genes were detected when a larger array was used. To minimize the impact of these factors, only genes that were differentially expressed overall are reported. Many genes associated with cell morphology, cellular development, and the immune and lymphatic system development and function were more highly expressed in resistant animals. The most significant GO terms associated with genes more highly expressed in resistant animals related to MHC class II function, and three MHC class II genes were confirmed to be more highly expressed in resistant animals (Table 2).

Genes with fatty acid metabolism related functions were also overrepresented in the list of genes more highly expressed in resistant animals. CYP4F2 is a member of the cytochrome P450 family, and catalyses the hydroxylation of leukotriene B4 (LTB4), a potent proinflammatory agent. LTB4 is known to be strongly induced in the intestine of rats infected with the nematode Nippostrongylus brasiliensis (24, 40), and these elevated levels can be associated with anaphylaxis in primed rats (35). Therefore, resistant animals many induce CYP4F2 to modulate LTB4 levels and reject parasites without inducing anaphylactic shock. HADHA and HADHB encode the two subunits of the mitochondrial trifunctional protein. The reason for the increased expression of these genes is unclear; however, gastrointestinal nematode infection is known to result in changes in feed intake and energy metabolism (21).

A number of genes involved in free radical scavenging were also more highly expressed in the duodenum of resistant animals. APP has been reported to increase superoxide generation in human neutrophils (5), while angiotensin II, the biologically active product generated by cleavage of AGT, is thought to stimulate intracellular reactive oxygen species (54). CYBB encodes a subunit of cytochrome B, a component of the microbicidal oxidase system of phagocytes, and is also involved in production of reactive oxygen species. The increased production of reactive oxygen species may contribute to the ability of resistant animals to respond to parasite infection. Paradoxically, the expression arrays showed both superoxide dismutase and catalase were also more highly expressed in resistant animals. The elevated expression of these genes may help prevent damage to the neighboring host tissue by the reactive oxygen species produced to resist nematode infection.

Genes more highly expressed in susceptible animals had a variety of functions, although many genes involved in gene regulation and DNA binding were more highly expressed. Five heat shock genes were more highly expressed in the duodenum of susceptible animals. These genes are known to be induced in response to stress and are involved in gastric mucosa protection (43). In addition six genes involved in chromatin assembly were more highly expressed in susceptible animals. These genes can regulate gene transcription through chromatin remodeling. A number of genes involved in protein chaperone activity were also more highly expressed in susceptible animals. The elevated expression of so many stress response genes in the duodenum tissue of the susceptible animals implies that these animals are altering gene and protein expression in response to high parasite loads.

We compared the differentially expressed genes identified in this study to those previously reported to be differentially expressed in these selection lines in the absence of nematode infection (29). No genes were found to be more highly expressed in resistant animals both pre- and postinfection; however, two genes were more highly expressed in susceptible animals pre- and postinfection. These genes were EVE1, which encodes SH3 domain protein D19, and TFF3, which encodes intestinal trefoil factor, a gene involved in mucosal protection and healing (31).

Three MHC class II genes, Ovar-DQA1, Ovar-DQB1, and Ovar-DRA, were more highly expressed in the duodenum of resistant animals. The increase in expression of DQA1 was due to both the higher frequency of DQA1 null alleles in the susceptible line and lower DQA1 expression in this line. Including the null allele, nine alleles of DQA1 were identified in this study and seven of these had been observed previously (57). The DQA1 allele frequency difference between resistant and susceptible animals approached significance when genetic drift was taken into account. However, another selection line of Romney sheep showed a significant null allele frequency difference between resistant and susceptible animals. In addition, the homozygous null allele of DQA1 was associated with susceptibility to intestinal nematodes in a population of commercial Perendale sires. Therefore an association exists between DQA1 status and resistance to internal parasites, at least in some populations. Association between resistance to internal parasites and markers in the MHC II locus has previously been demonstrated (11, 44); however, most prior studies have shown association with a marker in the Ovar-DRB1 gene. As there is a high level of linkage disequilibrium in the MHC region of many mammals including sheep (27, 38), an interaction could exist between polymorphisms at the DQA1 and DRB1 loci. Indeed, the observed effect at DQA1 could be due to the same polymorphism as that which causes the effect at DRB1.

The MHC II locus is known to be highly polymorphic and MHC variation contributes to an individual's resistance or susceptibility to immune challenge. Balancing selection, mate selection, frequency-dependent selection by parasites, and pathogens as well as heterozygote advantage have all been proposed as mechanisms to explain the high level of nucleotide diversity at the MHC locus (42). Heterozygosity in the DQA1 gene of humans has been shown to vary across populations from 0.36 to 0.90 with an average heterozygosity of 0.71 (52). In our study, the existence of null alleles at a high frequency in the population results in a large number of heterozygote animals that carry a single copy of the null allele. The overall heterozygosity (H) in the Perendale selection line population is estimated as 0.67; however, many of these animals carry only a single copy of DQA1. This would appear to put these animals at a selection disadvantage, as they can potentially recognize fewer foreign antigens than animals with two copies of the gene. DQA1 null alleles are reported to be present in sheep populations at a frequency of 27–45%, making it the most common DQA1 allele (20). The null allele must, therefore, provide animals with a selective advantage to be maintained in sheep populations. This advantage may be via linkage disequilibrium with the presence of a advantageous DQA3 allele, as animals lacking a DQA1 gene have been shown to carry a DQA3 gene (23).

Using ovine cDNA microarrays we identified almost 300 genes differentially expressed in duodenum tissue between lines of Perendale sheep genetically resistant or susceptible to internal parasites. Genes with a variety of different biological functions were differentially expressed although genes with MHC class II, reactive oxygen species scavenging, and smooth muscle function appeared to predominate the set of genes with increased expression in resistant animals. The expression analysis also identified a null allele of DQA1. This demonstrates the utility of expression studies in identifying genetic variants associated with quantitative traits. After accounting for genetic drift, we found the null allele to be associated with susceptibility to internal nematodes in some, but not all, populations, suggesting the null allele is not solely causal for susceptibility. The DQA1 gene is most likely in linkage disequilibrium with other alleles, and it is the combination of these alleles that determines resistance or susceptibility. The expression levels of each of the MHC proteins in vivo may also be critical in determining resistance status.


   ACKNOWLEDGMENTS
 
We thank Roger Wheeler, Wendy Bain, Chris Morris, Kevin Knowler, Mary Wheeler, Gordon Greer, and Nadia McLean for assistance with maintenance of the Perendale selection lines, animal handling, and slaughter. We thank Alan McCulloch for assembling the ovine ESTs and providing bioinformatic support. We thank Meredith Roberts-Thomson for genotyping the Romney selection line, Theresa Wilson and Dianne Hyndman for preparing the ovine microarray slides used, David Baird for carrying out the slide normalization procedure, and Robert McLaren for comments on the manuscript. Finally we thank the Ovita Consortium Ltd, which funded the study.


   FOOTNOTES
 
Address for reprint requests and other correspondence: J. C. McEwan, AgResearch Invermay Agricultural Centre, Private Bag 50034, Mosgiel, New Zealand (e-mail: john.mcewan{at}agresearch.co.nz).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

1 The online version of this article contains supplemental material. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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