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The possibility of positive selection for both F18 and stress resistant pigs opens new perspectives for pig
breeding
Annelies Coddens, Frank Verdonck, Martin Mulinge, Els Goyvaerts, Cora Miry, Bruno Goddeeris, Luc Duchateau, Eric Cox
To cite this version:
Annelies Coddens, Frank Verdonck, Martin Mulinge, Els Goyvaerts, Cora Miry, et al.. The pos- sibility of positive selection for both F18 and stress resistant pigs opens new perspectives for pig breeding. Veterinary Microbiology, Elsevier, 2007, 126 (1-3), pp.210. �10.1016/j.vetmic.2007.06.021�.
�hal-00532285�
Accepted Manuscript
Title: The possibility of positive selection for both F18+E.
coliand stress resistant pigs opens new perspectives for pig breeding
Authors: Annelies Coddens, Frank Verdonck, Martin Mulinge, Els Goyvaerts, Cora Miry, Bruno Goddeeris, Luc Duchateau, Eric Cox
PII: S0378-1135(07)00321-5
DOI: doi:10.1016/j.vetmic.2007.06.021
Reference: VETMIC 3744
To appear in: VETMIC Received date: 23-4-2007 Revised date: 19-6-2007 Accepted date: 25-6-2007
Please cite this article as: Coddens, A., Verdonck, F., Mulinge, M., Goyvaerts, E., Miry, C., Goddeeris, B., Duchateau, L., Cox, E., The possibility of positive selection for both F18+ E. coli and stress resistant pigs opens new perspectives for pig breeding, Veterinary Microbiology(2007), doi:10.1016/j.vetmic.2007.06.021
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Accepted Manuscript
The possibility of positive selection for both F18+E. coliand stress resistant 1
pigs opens new perspectives for pig breeding.
2 3
Coddens Annelies1, Verdonck Frank1, Mulinge Martin1, Goyvaerts Els4, Miry 4
Cora4, Goddeeris Bruno2, Duchateau Luc3, Cox Eric1*
5 6
1Laboratory of Veterinary Immunology, Faculty of Veterinary Medicine, Ghent 7
University, Salisburylaan 133, 9820 Merelbeke, Belgium 8
2Department of Biosystems, Faculty of Bioscience Engineering, K.U.Leuven 9
Kasteelpark Arenberg 30, B-3001 Heverlee, Belgium 10
3Department of Physiology and Biometrics, Faculty of Veterinary Medicine, 11
Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium 12
4Dierengezondheidszorg Vlaanderen, Hagenbroeksesteenweg 167, 2500 Lier 13
14
*Corresponding author:
15
Eric Cox 16
Tel: +3292647396 18
Fax: +3292647496 19
Manuscript
Accepted Manuscript
Abstract
20 21
F18+E. coliinfections causing post-weaning diarrhoea and/or oedema disease are 22
a major cause of economic losses in pig industry. To date, no preventive strategy 23
can protect pigs from F18+ E. coli infections. One of the most attractive 24
approaches to eliminate F18+ E. coli infections is the selection for pigs that are 25
resistant to F18+ E. coliinfections. However, this strategy was not believed to be 26
favourable because of reports of genetic association with the stress susceptibility 27
gene in the Swiss Landrace. To investigate this potential association more 28
thoroughly, 131 randomly selected Belgian hybrid pigs were genotyped for both 29
the F18+ E. coli resistance alleles (FUT1A) and the stress susceptibility alleles 30
(RYR1T) and their association was investigated by determining the linkage 31
disequilibrium. This linkage disequilibrium (LD = -0.0149) is close to zero and 32
does not differ significantly from 0 (likelihood ratio test 12 = 1.123, P = 0.29), 33
demonstrating no association between the FUT1A and RYR1T alleles. Furthermore, 34
only a small fraction (4.6%) of the Belgian pigs was found to be resistant to F18+ 35
E. coli infections. Unlike to what has previously been postulated, our results 36
suggest that selection for F18+E. coli resistant pigs might indeed be an attractive 37
approach to prevent pigs from F18+E. coliinfections.
38 39 40
Keywords: F18+E. coli (F107/86)/pig/ryr1/fut1/porcine stress syndrome 41
42
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1. Introduction
43 44
Many pig farms suffer from severe economical losses due to outbreaks of post- 45
weaning diarrhoea or oedema disease in young pigs. F18+ Escherichia coli 46
infections are a major cause of both diseases. A prerequisite for these infections is 47
the adherence of F18+E. coli to specific F18 receptors (F18R) on the porcine gut 48
epithelium, followed by colonization of the gut and excretion of entero- and/or 49
verotoxins leading to diarrhoea and/or oedema disease, respectively.
50
The prevalence of F18+ E. coli infections is estimated to be very high in Belgian 51
pig farms (up to 96%) (Verdonck et al., 2003). However, some pigs are found to 52
be resistant to colonization by F18+ E. coli due to lack of F18R expression.
53
Extensive mating studies revealed that the F18R status of pigs is genetically 54
determined with susceptibility dominating resistance (Bertschinger et al., 1993).
55
The F18R locus has been genetically mapped to the halothane linkage group on 56
porcine chromosome 6 and it was found that FUT1, encoding an 57
α(1,2)fucosyltransferase, is a candidate gene for this locus (Meijerink et al., 1997;
58
2000). Sequencing of the FUT1 gene revealed a polymorphism (G or A) at 59
nucleotide 307. The F18+ E. coli resistant pigs showed presence of the A 60
nucleotide on both alleles (FUT1A/A genotype), whereas pigs susceptible to F18+ 61
E. coli had either the heterozygous FUT1G/A or the homozygous FUT1G/G 62
genotype.
63
The HAL-locus, which also belongs to the halothane linkage group, determines 64
whether pigs are susceptible for malignant hyperthermia, an inherited 65
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neuromuscular disorder that is triggered by inhalational anaesthetics (halothane), 66
skeletal muscle relaxants (succinylcholine), as well as stress (Reik et al., 1983).
67
Therefore, this disease is often referred to as the porcine stress syndrome (PSS).
68
The underlying cause of this disease is the dysfunction of the Ca2+ release channel 69
(ryanodine receptor, RYR) of the skeletal muscle sarcoplasmatic reticulum 70
(O’Brien, 1986; Murayama et al., 2007), due to a mutation (C -> T) at nucleotide 71
1843, resulting in substitution of Cys615 for Arg in the ryanodine receptor (Fujii et 72
al., 1991).
73
In the present study, the prevalence of both the F18+ E. coli resistance allele 74
(FUT1A) and the malignant hyperthermia susceptibility allele (RYR1T) in the 75
Belgian pig population is estimated. Furthermore, the association of the FUT1A 76
alleles and the RYR1T alleles is investigated in order to address the possibility to 77
select for both stress resistance and F18+ E. coli resistance. Association of both 78
alleles would have the negative side-effect that an increase of the stress- 79
susceptibility allele frequency would occur when selecting for F18+ E. coli 80
resistant pigs. Our results will enable us to formulate advices on the usefulness or 81
potential drawbacks towards positive selection for F18+ E. coli resistant pigs in 82
the Belgian pig population.
83
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2. Materials and methods
84
2.1. Survey sampling 85
Blood samples from pigs out of 5 Belgian provinces were randomly collected by 86
veterinary practitioners and were sent to our laboratory by the regional veterinary 87
investigation centres. Twenty-one percent of the examined pigs were sows and 88
79% were fattening pigs. The pigs had different ages and were randomly selected 89
from both open and closed farms. The number of collected samples per province 90
was accorded to the number of pig-rearing farms present in each province (West- 91
Flanders n = 67, East-Flanders n = 33, Antwerp n = 18, Limburg n = 10, Flemish 92
Brabant n = 3). A total of 131 pigs from 36 different farms with a maximum of 5 93
pigs per farm were successfully genotyped for both the F18+ E. coli and stress 94
susceptibility genes. The selected pigs were not pure breed, but hybrid in nature.
95 96
2.2. DNA extraction 97
DNA was extracted from whole blood using the following procedure. One 98
hundred microliter blood was washed 3 times by adding 1 ml TE buffer (10 mM 99
Tris-HCl + 1 mM EDTA; pH 7.5) followed by centrifugation at 3800 g for 1 100
minute. Next, 200 µl K-buffer (50 mM KCl + 20 mM Tris-HCl + 2.5 mM MgCl2
101
+ 0.5% Tween20; pH 8.3) was added to the cell pellet, followed by 2 µl 102
proteinase K (Boehringer Mannheim) and this mixture was incubated at 56°C 103
during 1 hour. Inactivation of proteinase K was established by a 10-minute 104
incubation at 95°C. After centrifugation (3800 g, 1 minute), the supernatant 105
containing the genomic DNA was collected.
106
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107
2.3. Determination of the F18+E. coli genotype 108
PCR-RFLP based on the G->A mutation at nucleotide 307 of the FUT1 gene was 109
used for genotyping the pigs (Meijerink et al., 1997). Hereto, a fragment of the 110
FUT1 gene was amplified using specific primers (M307F: 5’- 111
CTTCCTGAACGTCTATCAAGACC-3’, M307R: 5’-CTTCAGCCAGGG-
112
CTCCTTTAAG-3’), followed by digestion with the CfoI restriction enzyme 113
(Promega) (Figure 1A).
114 115
2.4. Determination of the malignant hyperthermia genotype 116
In order to detect the C->T mutation on base pair 1843 of the RYR1 gene, a PCR- 117
RFLP-test was executed as described by Otsu and colleagues (1992). A fragment 118
of the RYR1 gene was amplified by PCR using specific primers (RYR1F: 5’- 119
TCCAGTTTGCCACAGGTCCTACCA-3’, RYR1R: 5’-ATTCACCGGAGT- 120
GGAGTCTCTGAG-3’). This resulted in a PCR product of 659 bp, which was 121
subsequently digested using Alw21I (HgiAI) (Fermentas) (Figure 1B).
122 123
2.5. Data analysis 124
Confidence intervals for the prevalence of the alleles were based on the binomial 125
distribution assumption. Heterogeneity of the prevalence of the alleles between 126
farms was studied by a generalized mixed model with a binomially distributed 127
error term and with farm as normally distributed random effect. The intraclass 128
correlation coefficient (ICC), a measure for the clustering of the allele in a farm, 129
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was obtained from this model and it was tested whether it differed significantly 130
from zero.
131
To investigate the association between the FUT1A alleles and the RYR1T alleles, 132
the linkage disequilibrium (LD) between the alleles was estimated and it was 133
further tested whether the LD differs from zero according to the methodology 134
discussed by Hill (1979), which is based on the likelihood ratio test. In addition, 135
the correlation coefficient between the two alleles was derived.
136
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3. Results
137 138
3.1. Prevalence of the F18+E. coli and stress susceptibility alleles 139
The prevalence of the F18+E. coli susceptibility genotypes FUT1G/G and FUT1G/A 140
in Northern Belgium was estimated to be 62.6% (confidence interval (CI) 95%
141
53.7-70.9) and 32.8% (CI 95% 24.9-40.9), respectively. The FUT1A/A genotype 142
referring to resistance to F18+E. coli infections was present in 4.6% (CI 95% 1.7- 143
9.7) of the cases (Figure 1). The frequency of the FUT1A and FUT1G allele was 144
0.21 and 0.79, respectively.
145
The prevalence of the stress resistance genotypes RYR1C/C and RYR1C/T in 146
Northern Belgium was found to be 67.2% (CI 95% 58.4-75.1) and 30.5% (CI 95%
147
22.8-39.2), respectively, whereas the stress susceptibility genotype RYR1T/T 148
accounted for 2.3% (CI 95% 0.5-6.5). Allele frequencies for the RYR1C and 149
RYR1T alleles were 0.81 and 0.19, respectively.
150
In addition, the heterogeneity over farms for theFUT1A and FUT1G alleles and the 151
RYR1C and RYR1T alleles was investigated. For the FUT1G and FUT1A alleles, no 152
differences could be found between the farms. However, when evaluating the 153
heterogeneity of the RYR1C and RYR1T alleles on Belgian farms, an intraclass 154
correlation coefficient of 0.76 (P < 0.0001) was found, revealing clustering of 155
RYR1C or RYR1T alleles on the Belgian farms.
156 157
3.2. Association between the F18+ E. coli resistance alleles and stress 158
susceptibility alleles 159
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Linkage disequilibrium refers to the non-random association of alleles at distinct 160
loci and is a reflection of the differences between the observed and expected 161
haplotype frequencies, supposed that the two loci are not independent (Pritchard 162
and Przeworski, 2001). In the present study, the linkage disequilibrium D equals - 163
0.0149, and does not differ significantly from 0 (12= 1.123, P = 0.29).
164
Accordingly, the correlation coefficient (r = - 0.096) showed no significant 165
correlation between both alleles. In Table I, it is demonstrated that 67% of the 166
FUT1A/A pigs (n = 6) showed the RYR1C/C genotype, while 33% of these pigs 167
showed the RYR1C/T genotype and none of these pigs had the stress susceptible 168
RYR1T/T genotype. For pigs with the heterozygous F18+ E. coli susceptible 169
genotype (FUT1G/A genotype, n = 43), it was found that 60% of the pigs were 170
homozygous stress resistant (RYR1C/C), 35% of the pigs were heterozygous stress 171
resistant (RYR1C/T) and 5% showed the stress susceptible genotypeRYR1T/T. For 172
pigs with the homozygous F18+E. coli susceptible genotype (FUT1G/G genotype, 173
n = 82), it was shown that 71% of the pigs generated the RYR1C/C genotype, while 174
28% had the RYR1C/T genotype and 1% had the RYR1T/T genotype.
175
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4. Discussion
176 177
The discovery of the halothane challenge test for phenotyping PSS pigs (Webb, 178
1980), even as the identification of the causal mutation in the porcine ryanodine 179
receptor (Fujii et al., 1991, Rempel et al., 1993) was highly important to decrease 180
the high prevalence of stress susceptibility in the Piétrain and Landrace pigs in the 181
late seventies (Van Zeveren et al., 1988). From that moment, the frequency of the 182
stress susceptible genotype in the Belgian landrace was monitored accurately and 183
decreased strongly. However, the majority of the Piétrain boars used for 184
production of fattening pigs is still stress-susceptible because of the relation of 185
this gene with excellent carcass meat quality (De Smet et al., 1996). The 186
combination of these boars with a homozygous stress resistant sow leads to a 187
mainly heterozygous stress resistant offspring. Belgian pig farmers are 188
encouraged to select for stress negative pigs by the ban on tranquilizers during 189
transportation and by the demand for stress-negativity of porcine meat products to 190
achieve the Belgian CERTUS-quality label.
191
Besides the prevalence, clustering of FUT1A or FUT1G alleles and RYR1C or 192
RYR1T alleles within farms was studied. For the RYR1 alleles, clustering of the C 193
as well as T alleles within farms could be found. This illustrates that testing of the 194
stress gene is nowadays common in pig industry. Some pig breeders prefer to 195
include the RYR1T allele in their herds in a controlled manner in order to obtain 196
optimal muscularity, whereas other pig farmers decide to exclude the stress 197
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susceptibility allele to avoid economic losses caused by pale, soft and exudative 198
meat.
199
The prevalence of the F18+ E. coli resistant genotype in the Belgian pig 200
population was estimated to be 4.6% and the allele frequency of the FUT1A allele 201
was 0.21. This is in accordance with data obtained by Meijerink et al. (1997) and 202
Vogeli et al. (1997), showing an FUT1A allele frequency of 0.21 after examination 203
of 455 animals. In addition, no clustering of the FUT1G and FUT1A allele was 204
found, indicating a random distribution of these alleles in the population. This 205
reflects absence of selection for F18+ E. coli resistant pigs in the studied pig 206
population. Furthermore, it is important to note that the prevalence of the F18+ E.
207
coli resistant genotype is strongly dependent on the breed. The Polish Zlotnicka 208
Spotted pigs, for instance, demonstrated a high occurrence (37.5%) of the 209
FUT1A/A genotype (Klukowska et al., 1999), whereas almost all native Chinese 210
breeds show complete absence of the FUT1A allele (Yan et al., 2003).
211
Unfortunately, the potential association with the stress susceptibility allele was 212
not assessed in these studies.
213
Analysis of the association between the stress susceptibility and F18+ E. coli 214
resistance alleles in the Belgian pig population by determining the linkage 215
disequilibrium demonstrated that both alleles are not associated. This is in contrast 216
with earlier findings of Meijerink et al. (1997), who revealed a 93% association 217
between FUT1A alleles and RYR1T alleles in the Swiss Landrace population and 218
probably therefore, the selection for F18+E. coli resistant pigs has not been taken 219
into practice until now. This difference between our data and the findings of 220
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Meijerink (1997) could be explained by the different population history of Swiss 221
Landrace pigs compared to Belgian pigs. It is possible that a founder effect in the 222
studied pedigree of Swiss Landrace pigs was causative for this high association.
223
Founder effects in pigs can arise because of the economically driven extensive 224
selection programmes, which reduce the effective population sizes strongly. In 225
Belgian commercial pigs for example, the effective population size is diminished 226
from thousands to less than 200 animals (Harmegnies et al., 2006).
227
Selection for FUT1A/Apigs implies that a positive selection for pigs deficient in the 228
FUT1 gene is carried out. These FUT1A/A pigs show decreased levels of FUT1 229
activity (Meijerink et al., 2000), even as an almost complete absence of 230
expression of histo-blood group antigens (HBGAs) on their intestinal epithelial 231
cells (Coddens et al., 2007). The necessity of expression of these HBGAs on the 232
intestinal epithelium for normal development and reproduction of pigs is not 233
studied yet, althoughFUT1 null mice and FUT2 null mice are proved to be viable, 234
healthy and fertile, indicating that the FUT loci and their fucosylated glycans are 235
not essential for normal development in mice (Domino et al., 2001).
236
Since no association between the F18+ E. coli resistance allele and the stress 237
susceptibility allele was found in the Belgian pig population, the authors believe 238
that selection for F18+ E. coli negative pigs can be taken into consideration.
239
Hereto, we recommend to establish small scale breeding programmes focussing 240
on selection for F18+E. coli negative pigs in order to extend the knowledge about 241
economical important traits such as reproduction rates and meat- or carcass 242
quality of F18+ E. coli resistant pigs. If no negative effects on these qualities are 243
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found, these studies could pave the way for a general positive advice on selection 244
for F18+ E. coli resistant pigs. Since no vaccine is available against F18+ E. coli, 245
this strategy to provide protection against F18+E. coliinfections could be of great 246
economical importance for the pig industry.
247 248 249
Acknowledgements
250
This research was funded by a PhD grant of the Institute for the Promotion of 251
Innovation through Science and Technology in Flanders (IWT-Vlaanderen).
252
F. Verdonck has a postdoctoral grant from the FWO Vlaanderen.
253
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Table and Figure legends
325
326
Figure 1: Detection of (A) the FUT1 G to A and (B) the RYR1 C to T mutation 327
by RFLP.
328
(A) Digestion of amplified FUT1 fragments with CfoI results in fragments of 241, 329
93 and 87 bp for the FUT1G/G genotype, whereas the FUT1G/A genotype generates 330
fragments of 328, 241, 93 and 87 bp and the FUT1A/A genotype creates fragments 331
of 328 and 93 bp. (B) Digestion of RYR1 PCR-product with Alw21I generates 332
fragments of 524, 358, 166 and 135 bp for the RYR1C/T genotype, while the 333
RYR1C/C genotype results in fragments of 524 and 135 bp and the RYR1T/T 334
genotype generates fragments of 358, 166 and 135 bp.
335 336
Table I: Correlation between F18+E. coli and stress susceptibility genotypes 337
338 339 340 341 342 343 344 345
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Figures
(A) FUT1 genotype (B) RYR1 genotype
Figure 1: Detection of (A) the FUT1 G to A and (B) the RYR1 C to T mutation by RFLP.
(A)Digestion of amplified FUT1 fragments with CfoI results in fragments of 241, 93 and 87 bp for the FUT1G/G genotype, whereas the FUT1G/A genotype generates fragments of 328, 241, 93 and 87 bp and the FUT1A/A genotype creates fragments of 328 and 93 bp.
(B) Digestion of RYR1PCR-product with Alw21I generates fragments of 524, 358, 166 and 135 bp for the RYR1C/T genotype, while the RYR1C/C genotype results in fragments of 524 and 135 bp and the RYR1T/T genotype generates fragments of 358, 166 and 135 bp.
GA AA GG
328 bp 241 bp 93 bp 87 bp GG GA AA
524 bp 358 bp
166 bp 135 bp CT CC TT
Figure
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Tables
Table I: Correlation between F18+E. coliand stress susceptibility genotypes
RYR1
FUT1 CC(n =88) CT(n = 40) TT(n = 3)
58 23 1
26 15 2
GG (n = 82) GA (n = 43)
AA (n = 6) 4 2 0
Table
