Association between microsatellite markers of sheep chromosome 5 and faecal egg counts

Small Ruminant Research 46 (2002) 97–105

Association between microsatellite markers of sheep chromosome 5 and faecal egg counts M.V. Benavides a,∗ , T.A. Weimer b , M.F.S. Borba a , M.E.A. Berne c , A.M.S. Sacco a a

b

EMBRAPA Pecuária Sul, P.O. Box 242, CEP 96401-970, Bagé, RS, Brazil Department of Genetics, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil c Universidade Federal de Pelotas, Pelotas, RS, Brazil Accepted 30 July 2002

Abstract This study analyses the associations between alleles at seven microsatellite markers located at the sheep chromosome 5—where the interleukin IL-3, IL-4 and IL-5 genes were mapped—and faecal egg counts (FEC) in two different flocks. The associations were analysed by estimating the average excess of alleles at each microsatellite marker on FEC. Three of the studied markers (CSRD2138, OarAE129 and TGLA176) showed significant associations with FEC in the Corriedale flock. Seven alleles at these microsatellite loci were associated with FEC and their effects ranged from −28 to +20%, relative to the FEC population mean. On the other hand, four alleles at CSRD2138 and TGLA176 microsatellite markers had significant associations with FEC in the Polwarth mob, with effects varying from −22 to −5%, relative to the FEC population mean. All microsatellites investigated presented high diversity in both flocks. Some alleles were breed specific. The CSRD2138∗ A allele was the only marker associated with consistent reductions in FEC for both breeds. These results indicate that markers or genes nearby the interleukin genes, such as the CSRD2138, might enhance host resistance to internal parasites in sheep by probably increasing effector cells and antibody responses. © 2002 Published by Elsevier Science B.V. Keywords: Disease association; Faecal egg counts; Gastrointestinal parasites; Microsatellites; Sheep

1. Introduction Gastrointestinal parasites are responsible for important economic losses in livestock. Increases in production costs, anthelmintic resistance and consumer’s concern about drug residues in animal products are a worry for the sheep industry as a whole. Selection for increasing resistance to infection or resilience to the effects of infection in sheep are regarded as alternatives to reduce the problem (McEwan, 1994; ∗ Corresponding author. Tel.: +55-53-242-8499; fax: +55-53-242-8499. E-mail address: [email protected] (M.V. Benavides).

Bisset and Morris, 1996). An increase in the number of resistant individuals will also benefit the entire flock as it will likely reduce pasture contamination and promote survival of animals with intermediate susceptibility levels. Sheep resistance to gastrointestinal parasites is usually based on faecal egg counts (FEC) and is genetically determined. Heritability estimates for FEC lay between 0.14 and 0.44 (Piper, 1987; Watson et al., 1986; Baker et al., 1991; McEwan et al., 1992; Morris et al., 1997) and guarantee low to moderate genetic progress for the trait. Although FEC and some immunological parameters (Douch et al., 1995; Bisset et al., 1996; Shaw et al., 1999) can be used

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as potential criteria for selecting sheep for resistance to gastrointestinal parasites, the length and costs of field experiments to phenotypically assess individuals are the major drawbacks of these approaches. The finding of a genetic marker associated with resistance/susceptibility to nematodes would allow earlier selection of desirable genotypes. Investigations on these genes would also help researchers to uncover the immunological mechanisms responsible for resistance to gastrointestinal parasites. In this sense the main research focus has been on genes of the major histocompatibility complex (MHC), which are involved in immunological induction and regulation processes and have been particularly targeted due to their high polymorphism. Schwaiger et al. (1995) found an association between a DRB1 allele and FEC reductions of 58-fold in Scottish Blackface lambs naturally infected with Teladorsagia circumcincta. Later, Buitkamp et al. (1996) found that some alleles from MHC Class I and DY (Class II) loci were associated with 8- and 218-fold decrease in FEC, respectively, in that same flock. Outteridge et al. (1985) found associations between resistance to Trichostrongylus colubriformis and MHC Class I in divergent lines of sheep selected for and against response to this parasite. However, the relationship between this gene and sheep selected for low FEC was not observed in a later study (Outteridge et al., 1986). Similarly, Cooper et al. (1989) and Crawford et al. (1997) found no effect of MHC on the susceptibility of sheep to gastrointestinal parasites. Nonetheless, it is well known that immunity to parasite antigens depends upon intricate immunological mechanisms, therefore it would be unlikely that host resistance could be affected by a major gene. However, studies associating non-MHC immunological mechanisms with resistance to nematode infection have been scarcely done (Clarke et al., 2001; Coltman et al., 2001). Immunological studies have shown that young lambs were unable to build immunological responses against Haemonchus contortus due to weak Th2 responses (Schallig, 2000). Th2 -type cytokines, especially high IL-4 mRNA expression levels in the gastrointestinal lymphatic tissue, were also important for the immune responses in sheep genetically resistant to T. colubriformis after natural challenge (Pernthaner et al., 1997). Investigations in mice have indicated that mastocytosis, eosinophilia and high IgE levels, which

normally characterise Th2 responses and are IL-3, IL-4 and IL-5-dependent, are the main mechanisms for host defence against parasitic gastrointestinal nematodes (Finkelman et al., 1991; Finkelman and Urban, 1992; Finkelman et al., 1997). In sheep, these cytokines are located at chromosome 5 (http://www.thearkdb. org). The objective of this study was to verify possible associations between seven microsatellite markers located nearby these cytokines and FEC in weaned lambs from two different flocks challenged under same natural conditions.

2. Materials and methods 2.1. Populations Unrelated weaned ewe lambs of approximately 7 months of age of the Corriedale (n = 48) and Polwarth (n = 82) breeds from EMBRAPA/CPP Sul, Bagé, Rio Grande do Sul, Brazil (54◦ 23 W and 30◦ 47 S) were used in the trial. Selection for reduced FEC was never attempted although efforts to select for wool production were made in the past. The animals were raised as a single flock and challenged according to the methodology described in McEwan (1994). Briefly, animals kept on naturally contaminated paddocks were drenched to zero faecal egg counts. Faecal samples were randomly collected on 5% of the individuals on a weekly basis. When FEC ranged between 800 and 1500, all animals were individually sampled and drenched again to resume a successive challenge. This procedure was repeated two more times in early autumn (from March to May). The extremely wet (total monthly rainfall ranged from 150.3 to 235.8 mm) and hot (the monthly averages of extreme temperatures ranged from 19.8 to 27.4 ◦ C) climatic conditions during these months resulted in a rapid parasitic re-infestation of the lambs. The number of nematode eggs g−1 of faeces was determined by using a modified McMaster method (Whitlock, 1948). Briefly, 2 g of faeces are diluted in 58 ml of NaCl hypersaturated solution and 0.3 ml of the mixed solution is transferred to a McMaster chamber. The total number of eggs is multiplied by 100 to obtain FEC.

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Table 1 Primer sequences and references of the microsatellite markers (MS) used MS marker CSRD2134 CSRD2138 McM108 OarAE129 RM006 TGLA137 TGLA176

Primer sequences (5 –3 )

Reference

AAG GAC CTA CTA TTT AGC ACA GGG AGT GAT ACT TCA CAC ATG CCT ATG AGA TGC TAT TCC AAA CAC AGT CCC CAT GGG GTC ACA AAG AGT TGG ACA CTT AAG CCA GGT GTT TCA AAG ATG TCT CTT TTT CTC TCT CTC TCT GAA ACA AAT CCA GTG TGT GAA AGA CTA ATC CAG GTA GAT CAA GAT ATA GAA TAT TTT TCA ACA CC CTA CAA TAT CTG GTC ACT GGA GAT CAC CAT ATT TAT GAG ATG G GTT GAC TTG TTA ATC ACT GAC AGC C CCT TAG ACA CAC GTG AAG TCC AC GCT TTA AGA ACT GCT CTC AAG GCA C CTG GGT GAG AAA TGG GTA GTA GG

Davies et al. (1996) Drinkwater et al. (1997) Smith et al. (1995) Penty et al. (1993) Kossarek et al. (1993) Georges and Massey (1992) Georges and Massey (1992)

2.2. Genotyping

2.3. Statistical analyses

Lambs were blood sampled by venipucture for DNA extraction (Miller et al., 1988). DNA samples were amplified with the microsatellite markers described in Table 1 on a Perkin-Elmer 2400® thermocycler machine using the PCR conditions described in the cited references. Amplified PCR products were run in 1.2-mm thick non-denaturing 10% polyacrylamide gels, except for TGLA176 where 13% polyacrylamide gels were used. Fragment visualisation was done after dying gels on 0.5 ng/ml ethydium bromide (Lahiri et al., 1997). Molecular weight markers were prepared by digesting pBR322 and φX174/Hinf I vectors with restriction endonucleases. Polyacrylamide gels were then photographed with a Kodak DC120 camera and fragment sizes analysed by using the Kodak Digital Science 1D® software. To verify whether associations between alleles and FEC in the Corriedale population were not flock specific, microsatellite markers that showed significant effect in the Corriedale population were tested on a second flock (Polwarth). This Polwarth population had two important characteristics: (a) it was field challenged with the Corriedales as a single mob, and (b) it had different allelic frequencies from the Corriedales thus providing a reference flock to validate the results found in this latter flock.

Association between each microsatellite marker and FEC was determined by the average excess analysis according to Templeton (1987). The average excess for each allele within each marker was estimated according to the formula: k
ai = [fik /pi ][yik − y], i = 1, . . . , k k=1

where, yik is the phenotypic mean of each ik genotypic class, y is the phenotypic mean of the population, fik is the frequency of the ik genotype and pi the frequency of allele i. Significance of the average excesses were compared against a theoretical sampling distribution generated by 1000 permutations where phenotypic values were randomly allocated over the genotypes using the random command of the Minitab V.10® software (Haviland et al., 1997; Andrade et al., 2000). The analyses were run after logarithmic transformation of FEC values [log10 (FEC + 25)], where the value of 25 was arbitrarily defined in order to include 0 (zero) FEC values in the analyses. Results were expressed as percentage relative to the population mean. All statistical analyses were run independently per allele within marker and population. The allele frequencies between populations were compared by using a χ 2 -test. Because small expected

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Table 2 Descriptive statistics for the mean FEC and mean log FEC (mean± S.D.) of the three successive challenges made in the two populations Population

FEC (eggs g−1 ) Mean ± S.D.

Range

Corriedale Polwarth

3317 ± 1736 2714 ± 1005

567–10,366 733–7266

Mean log FEC 3.31 ± 0.29 3.27 ± 0.17

P ≥ 0.05.

frequencies occurred in all comparisons, the P values were estimated by using the Monte Carlo solution of Roff and Bentzen (1989). Polymorphism information content (PIC) was calculated to determine the within-flock genetic variability (Botstein et al., 1980).

The polymorphic information content (PIC) and number of alleles for each of the markers studied are presented in Table 3. Both populations showed high genetic diversity for the majority of markers Table 4 Allele frequencies (%) for the CSRD2138, OarAE129 and TGLA176 markers, FEC population means (eggs g−1 ) and total number of alleles (N) in each population Marker

Allele

CSRD2138

A B C D E F G H I

3. Results The overall FEC mean (±standard deviation) for each population, and its range, as well as log FEC mean (±S.D.) are shown in Table 2. There was no significant difference (P ≥ 0.05) in the log FEC mean between populations. Faecal egg counts tended to be a highly variable trait. Cultured eggs showed that H. contortus was the most predominant helminth species during the experimental challenge, accounting for 65% of the parasites on the three samplings, followed by Strongyloides papillosus (28%), Teladorsagia spp. (4%) and Cooperia spp. (3%) which is in agreement with epidemiological studies previously made in the region (Pinheiro, 1987). Table 3 Polymorphic information content (PIC) and number of alleles for each marker in each population Marker

Allele

PIC

Corriedale CSRD2134 CSRD2138 McM108 OarAE129 RM006 TGLA137 TGLA176

4 8 12 9 3 6 11

0.56 0.76 0.87 0.80 0.29 0.52 0.86

Polwarth CSRD2138 OarAE129 TGLA176

9 9 11

0.81 0.83 0.87

Population mean ± S.D. A B C D E F G H I J

N

1.43∗ 2.14 7.14 15.71 20.00∗ 18.57 21.43 10.71∗ 2.86

3.26 4.35 27.17 15.22 5.43 5.43∗ 16.30 20.65 2.17∗ 0.00 3279 ± 1751

N

Population mean ± S.D.

Polwarth

3.33∗∗ 1.11 20.00 4.44 30.00∗ 20.00 17.78 3.33 0.00

90

Population mean ± S.D. TGLA176

Corriedale

3364 ± 1760

N OarAE129

Allele frequencies (%)

92 A B C D E F G H I J K L

19.05 19.05 13.10∗∗ 5.95 0.00 2.38 4.76 9.52 3.57∗ 10.71 9.52∗ 2.38 3338 ± 1754 84

2835 ± 1407 140 14.29 5.71 16.43 20.71 8.57 0.00 5.00 20.00 7.86 1.43 2783 ± 1441 140 6.96∗ 13.92 22.15 6.96 3.16 0.00 6.96 9.49 10.13 10.13 7.59 2.53 2748 ± 1412 158

Italicised values highlight population-specific alleles. ∗ P < 0.05 (alleles with significant associations with FEC). ∗∗ P < 0.01 (alleles with significant associations with FEC).

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Fig. 1. Allelic effects of CSRD2138 on FEC average in the Corriedale and Polwarth flocks (population mean ± standard deviation: 3364 ± 1760 and 2835 ± 1407, respectively). Significant average excesses are indicated with .

tested. Most of the PIC values were high, a reflection of the high number of alleles and their equilibrated frequencies within each marker. The exception was TGLA137 which, despite having six alleles, presented a PIC value lower than that for CSRD2134 (four alleles). One particular TGLA137 allele showed a remarkable high frequency (62%; non-tabulated) in the Corriedale population, which likely reduced this particular marker’s diversity. Table 4 presents the allele distributions for the CSRD2138, TGLA176 and OarAE129 microsatellites in both populations as well as their FEC mean values.

For the other non-significant markers, allele frequencies were not tabulated but are available on request. Allele frequencies significantly differed between the two populations studied. Some alleles such as CSRD2138/I, OarAE129/F, OarAE129/J, TGLA176/E and TGLA176/F were population specific. For the Corriedale population, the average excess analyses indicated that CSRD2138∗∗ A, OarAE129∗ I, OarAE129∗ F and TGLA176∗∗ C were associated with significant reductions of 28, 20, 16 and 10% relative to the FEC population mean, respectively (P < 0.05; Figs. 1–3). CSRD2138∗ E, TGLA176∗ K and

Fig. 2. Allelic effects of OarAE129 on FEC average in the Corriedale and Polwarth flocks (population mean ± standard deviation: 3279 ± 1751 and 2783 ± 1441, respectively). Significant average excesses are indicated with .

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Fig. 3. Allelic effects of TGLA176 on FEC average in the Corriedale and Polwarth flocks (population mean±standard deviation: 3338±1754 and 2748 ± 1412, respectively). Significant average excesses are indicated with .

TGLA176∗ I were associated with FEC values of 4, 9 and 20% above the population mean, respectively. The other markers showed no significant effects on FEC. In the Polwarth population, CSRD2138∗ A, CSRD2138∗ H, TGLA176∗ A and CSRD2138∗ E carriers were associated with reductions of 22, 10, 9 and 5% in FEC, relative to the population mean, respectively (P < 0.05). The OarAE129 marker showed no significant effect on FEC. Interestingly, CSRD2138∗ E showed opposite significant effects for both populations. Haplotype analysis was not attempted due to the low number of animals in each combination of genotypes.

4. Discussion The high infestation levels observed in the experimental lambs are typical of Southern Brazil sheep production systems and stress the need of finding alternatives for controlling small ruminant parasitism, such as selection of more resistant individuals. According to Bisset and Morris (1996) and Douch et al. (1996), selection for resilience, the ability of sheep to withstand the pathogenic effects of parasite infection, could be more important than selection for resistance. Nonetheless, Alberts et al. (1987) had observed that resistance to H. contortus had positive genetic correlation with resilience. Therefore, at least in regions where this helminth is the predominant species, selection for increased resistance could be a better strategy

to lower the levels of parasite burdens in the pasture, reduce re-infestation and production losses. Other selection criteria such as eosinophil, mast cell counts, and antibody titres have been suggested (Douch et al., 1996). However, selection based on faecal egg counts is relatively inexpensive compared to other criteria, easy to sample and reflects the parasitic burden at the time of sampling. Despite the fact that selection for low FEC sheep has negative genetic correlations with growth rate and wool growth (McEwan et al., 1992), there is general agreement among sheep farmers that FEC is a worthwhile criterion because it could reduce production costs by decreasing the number of drenches. A survey has shown that sheep raised in Southern Brazil are drenched, on average, nine times a year (Echevarria et al., 1996) and the increasing cost of the parasitic control is high enough to hamper sheep production in this region. The high phenotypic variation for faecal egg counts and genetic diversity of the flocks studied allowed the detection of significant associations between FEC and nine alleles of the CSRD2138, TGLA176 and OarAE129 markers. The magnitude of the effects observed in the two populations varied from −28 to +20% relative to their FEC averages. These were moderate results when compared to the effect of some alleles of the MHC Class I, DY (Class IIb subregion) and DRB1 genes reported by Schwaiger et al. (1995) and Buitkamp et al. (1996) that ranged from 8- to 218-fold FEC reduction in lambs predom-

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inantly infected with T. circumcincta. Despite being moderate, these values are relevant and can reduce the negative impact on production caused by high parasitic infestations of H. contortus. Some alleles showed either significant associations with FEC in one flock but not on the other, or even opposite FEC effects in the two flocks as in the case of CSRD2138∗ E. These alleles will have limited utilisation for selection purposes in the ovine species as a whole. However, CSRD2138∗ A was the only allele associated with consistent reductions in FEC average at both populations (at least 22%) and it is a promising marker for resistant genotype in sheep. This relationship was observed regardless the low allelic frequency of CSRD2138∗ A in both populations, indicating that there is chance for progress on FEC reduction by selecting carriers since it is an allele far from being fixed. It is worth noting that CSRD2138 and IL-3, IL-4, and IL-5 genes are closely situated in sheep chromosome 5 (Maddox et al., 2001). Considering that IL-3, IL-4 and IL-5-dependent immunological mechanisms are chief processes in mice (Finkelman et al., 1991; Finkelman and Urban, 1992; Finkelman et al., 1997) and sheep (Pernthaner et al., 1997; Gill et al., 2000; Schallig, 2000) defences against gastrointestinal parasites, it could be hypothesised that CSRD2138 up-regulates the level of transcription of interleukins 3, 4, and 5 leading to better responses against internal parasites. Despite being unlikely that a microsatellite marker (DNA repeats) could be directly responsible for differences in phenotypes, DNA repeats are mainly situated at promoter regions and are able to shift the DNA molecule conformation to Z-type DNA. Changing conformation to Z-type DNA will favour RNA polymerase binding and increase mRNA transcripts of genes situated close to the DNA repeats (Comings, 1998). But to confirm the genetic link between CSRD2138 and the cited interleukins, further confirmation of these findings will have to be done using linkage analysis on a flock especially designed for this purpose.

5. Conclusions Significant associations between alleles of the CSRD2138, TGLA176 and OarAE129 microsatellite markers and faecal egg counts were observed in

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two commercial flocks where sheep were naturally parasitised, predominantly with H. contortus. Among these alleles, CSRD2138∗ A was the only one to be associated with FEC in the two unrelated and unselected flocks, regardless of the fact that allele frequencies significantly differed between both. Reductions of about 22% in FEC were associated with this specific allele in both flocks.

Acknowledgements We thank Dr. Noelle Cockett (University of Utah) for kindly donating the primers used in the experiment. The authors also thank FAPERGS, IICA/PRODETAB, FINEP and CNPq for funding the project and financing scholarship for MVB.

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