Cox, Eric

Duchateau, Luc

Goddeeris, Bruno

Rasschaert, Kristien

VERDONCK, F

English

Ghent University Academic Bibliography

www.elsevier.com/locate/vetmicVeterinary Microbiology 123 (2007) 249–253Short communicationScreening of pigs resistant to F4 enterotoxigenicEscherichia coli (ETEC) infectionK. Rasschaert a, F. Verdonck a, B.M. Goddeeris a,b, L. Duchateau c, E. Cox a,*aLaboratory of Immunology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, BelgiumbDepartment of Biosystems, Faculty of Bioscience Engineering, KULeuven, Kasteelpark Arenberg 30, B-3001 Leuven, BelgiumcDepartment of Physiology and Biometrics, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, BelgiumReceived 13 November 2006; received in revised form 13 February 2007; accepted 19 February 2007AbstractThe present study analysed quantitatively themucin 4 polymorphism for determing the F4ac/ab receptor status of a total of 63pigs by comparing it with the in vitro villous adhesion assay. The probability of a susceptible genotype for the mucin 4 increasessignificantly with increasing F4ab or F4ac ETEC adhesion per 250 mm villi (P = 0.029 for F4ab, P = 0.030 for F4ac), with theodds ratio for each unit increase of F4ab or F4ac equal to, respectively, 1.036 (95%CI [1.004–1.069]) and 1.018 (95%CI [1.002–1.034]). In the phenotypic in vitro villous adhesion test, a cut-off value of 5 bacteria was chosen as a criteria for the distinctionbetween an F4R positive and F4R negative pig. The sensitivity and specificity for the in vitro villous adhesion test, with thegenotyping test for mucin 4 as golden standard, is 100% and 24%, respectively, for F4ab as well as F4ac. Absence of adhesion ofF4ac and F4ab ETEC to the villous brush borders was not associated with genotypic resistance suggesting that there is at leastone other receptor for F4ab/ac Escherichia coli. As a consequence, not only mucin 4 gene polymorphism but also expression ofthese other receptor(s) has to be included in a screening assay for F4ac/ab receptor negative pigs.# 2007 Elsevier B.V. All rights reserved.Keywords: Mucin 4; F4ab/ac ETEC; In vitro adhesion assay1. IntroductionInfections with enterotoxigenic Escherichia coli(ETEC) that express the F4ab or F4ac fimbriae, causediarrhea and mortality in neonatal and newly weanedpiglets. These pathogens bind with their F4ab/ac* Corresponding author. Tel.: +32 9 264 73 96;fax: +32 9 264 74 96.E-mail address: eric.cox@ugent.be (E. Cox).0378-1135/$ – see front matter # 2007 Elsevier B.V. All rights reserveddoi:10.1016/j.vetmic.2007.02.017fimbriae to F4 specific receptors (F4R) on the brushborder of small intestinal enterocytes, resulting incolonization and a secretory watery diarrhea (Gyles,1994). These receptors are not present in every pig andtheir absence causes resistance to F4 ETEC induceddiarrhea (Sellwood et al., 1975). Their expression isgenetically determined and inherited in a dominantway (Bijlsma and Bouw, 1987; Gibbons et al., 1977).The gene underlying resistance to F4ab/ac ETEChas been assigned by linkage analysis to porcine.K. Rasschaert et al. / Veterinary Microbiology 123 (2007) 249–253250chromosome 13 (Gue´rin et al., 1993), whereas theF4ad ETEC receptor is localized on another chromo-some (Peelman, 1999). For this reason, we did notfocus on the F4ad ETEC in our study. Moreover, apolymorphism in the mucin 4 gene has been linked tothe F4ab/ac ETEC adhesive phenotype and not for theF4ad ETEC adhesive phenotype. A DNA marker-based test has been developed to allow genotyping forF4ab/ac ETEC resistance/susceptibility (Jørgensenet al., 2004). Three different genotypes were observedand were called resistant (RR), susceptible hetero-zygote (SR) and susceptible homozygote (SS).Breeding programs with F4R negative pigs couldresult in prevention of F4ab/ac induced ETEC diarrheain the pig population. Therefore, an efficient screeningassay for genetic F4ab/ac resistance is needed. Thecurrent analysis for F4ab/ac susceptibility is based onan in vitro adhesion assay using villi (Girardeau, 1980)or brush border epithelial cells (Sellwood et al., 1975).The in vitro villous adhesion assay is performed byincubating small intestinal villi with F4ab or F4acETEC. Then, villi are examined by phase-contrastmicroscopy and the adhesion of bacteria is evaluatedquantitatively. Since the adhesion assay eitherdemands major intestinal surgery or slaughter of thepig, it makes it difficult to incorporate it into breedingprograms. A DNA-based test is more preferablebecause it allows precise genotyping of living animals.The aim of the present study was to perform aquantitative analysis for the mucin 4 polymorphismand the in vitro villous adhesion test for 63 pigs from31 different litters to investigate the relationshipbetween the genotype test for mucin 4 on the one handand the villous adhesion test for F4ac and F4ab on theother hand.2. Materials and methods2.1. The in vitro villous adhesion assay for F4RcharacterizationSixty-three pigs (with a age difference of 2 months)from 31 different sows and 14 different boars (BelgianLandrace  Pie´train, 20 pigs; Belgian Landrace Dutch Landrace, 12 pigs; Large White  Pie´train, 31pigs), on three different pig farms in Flanders weretested. In order to determine the presence or abscenceof the F4R on the brush border of small intestinalvillous enterocytes, an in vitro villous adhesion assaywas performed as described by Van den Broeck et al.(1999). Briefly, a 15 cm long intestinal segment wasexcised of the mid jejunum at the moment of slaughter.The segment was washed twice with PBS and oncewith Krebs–Henseleit buffer (0.12 M NaCl, 0.014 MKCl, 0.001 M KH2PO4 and 0.025 M NaHCO3adjusted to pH 7.4) containing 1% (v/v) formaldehydeat 4 8C. Subsequently, the villi were scraped off withglass slides and washed four times in Krebs–Henseleitbuffer without formaldehyde whereafter they wereresuspended in PBS supplemented with 1% (w/v) D-mannose (Fluka, Sigma–Aldrich, Bornem, Belgium)to prevent adhesion by type 1 pili. Subsequently4  108 F4ac bacteria (strain GIS 26, serotypeO149:K91, F4ac+, LT+STa+STb+) or F4ab bacteria(strain G7, serotype O8:K87:F4ab+, LT+) were addedto an average of 50 villi in a final volume of 0.5 ml andwere incubated at room temperature for 1 h whilegently shaking. The adhesion of bacteria wasevaluated by counting the number of bacteria adheringalong a 50 mm villous brush border length at 20randomly selected places with a phase-contrastmicroscope at a magnification of 600. Adhesion ofmore than 5 bacteria per 250 mm villous brush borderlength was noted as positive (Cox and Houvenaghel,1993). Each villous adhesion test included somenegative controls. First, the villi were checked beforeaddition of F4ab/ac bacteria to exclude adheringbacteria from the intestinal flora. Second, F4 bacteriawithout the adhesin (FaeG) were added. Finally, strainE57 (serotype O138: K81, STa+STb+), bacteriawithout fimbriae, was added to the villi.2.2. The DNA marker-based testThe DNA marker-based test was performed asdescribed by Jørgensen et al. (2004). It relies on a XbaIpolymorphism in intron 7 of the porcine mucin 4 gene.The PCR-RFLP assay was performed on genomicDNA from pigs in a total volume of 20 ml using 1Supertaq buffer (HT Biotechnology Ltd., Cambridge,England), 2 mM MgCl2, 200 mM of each dNTP,1.0 mM of each primer: 50-GTGCCTTGGGTGA-GAGGTTA-30/50-CACTCTGCCGTTCTCTTTCC-30,and 0.25 units Supertaq (HT Biotechnology Ltd.,Cambridge, England). Thermocycling was performedK. Rasschaert et al. / Veterinary Microbiology 123 (2007) 249–253 251using 15 min initial denaturation at 95 8C andsubsequently 95 8C for 15 s in the additional cycles.The annealing temperature was 65 8C and theextension was carried out at 72 8C for 1 min for 35cycli. The PCR product obtained from pig genomicDNA is 367 bp and 5.5 ml of the PCR products is usedfor XbaI digest as recommended by the supplier (NewEngland Biolabs, MA, USA). The resistant allele (R)is indigestible by XbaI, whereas the susceptible allele(S) is digested into 151 bp and 216 bp fragments. As acontrol for the XbaI digestion, we used a plasmid withone restriction site for XbaI.2.3. Statistical analysisThe relationship between the genotype test formucin 4 on the one hand and the villous adhesion testfor F4ac and F4ab on the other hand was investigatedthrough a logistic regression model including sow asrandom effect. The receiver operating characteristic(ROC) curve was made to investigate the effect ofFig. 1. Box-and-whisker plots of the in vitro villous adhesion test for F4ab(SR/SS) genotype of 63 pigs.The box indicates the 25th and 75th percentwhiskers are the extreme values. One point represents one pig.different cut-off values for F4ab and F4ac (distinctionbetween F4R+ and F4R) with respect to sensitivityand specificity. Repeatability of the in vitro villousadhesion test for F4ab and F4ac was studied through amixed model with pig as random effect.3. Results and discussionSixty-three pigs were tested in the in vitro villousadhesion assay and were genotyped for the XbaIpolymorphism in the mucin 4 gene. We tested thereliability of the genotyping by checking all theresistant genotypes by DNA sequencing on the PCRproducts. Results are presented for resistant (RR) orsusceptible (SS, SR) animals using a box-and-whiskerplot (Fig. 1). The box indicates the 25th and 75thpercentiles and the central line is the median. Thepoints at the ends of the whiskers are the extremevalues. Six of the 63 pigs had less than 5 F4ac or F4abbacteria adhering per 250 mm villous brush borderand F4ac fimbriae for the resistant genotype (RR) and the susceptibleiles and the central line is the median. The points at the ends of theK. Rasschaert et al. / Veterinary Microbiology 123 (2007) 249–253252Fig. 2. The receiver operating characteristic (ROC) curve, obtainedby plotting the false positive rate (1-specificity) versus the truepositive rate (sensitivity) for various cut-off values of F4ab (full line)or F4ac (dotted line). The numbers in the plot refer to specific cut-offvalues. For instance, for a cut-off value of 48 for the F4ac test, thesensitivity and specificity are 66% and 40%, respectively, whereasfor a cut-off value of 45 for the F4ab test, the sensitivity andspecificity are 76% and 40%, respectively.length in the in vitro villous adhesion assay and had aresistant (RR) genotype. Fifty-seven pigs had morethan 5 F4ac or F4ab bacteria adhering per 250 mmvillous brush border length. Nineteen of the 57 pigshad a RR genotype and the number of F4ab adheringbacteria ranged between 8 and 95. The number of F4acbacteria in the 19 pigs with a RR genotype rangedbetween 5 and 125. Thirty-eight pigs had at least onecopy of the susceptible haplotype (SR or SS). Thenumber of F4ab adhering bacteria for these 38 pigsranged between 6 and 105 with a median of 61. Thenumber of F4ac adhering bacteria ranged between 6and 137 with a median of 61.The probability of a SR/SS genotype for mucin 4increases significantly with increasing F4ab ETEC per250 mm villi (P = 0.029), with the odds ratio for eachunit increase of F4ab equal to 1.036 (95% CI [1.004–1.069]). For F4ac, the probability of a SR/SS genotypefor mucin 4 increases significantly with increasingF4ac ETEC per 250 mm villi (P = 0.030), with theodds ratio for each unit increase of F4ac equal to 1.018(95% CI [1.002–1.034]).The sensitivity and specificity of the in vitro villousadhesion test for F4ab and F4ac bacteria with thegenotyping test for the mucin 4 as golden standard isrepresented in Fig. 2. The ROC curve is obtained byplotting the false positive rate (1-specificity) versusthe true positive rate (sensitivity) for various cut-offvalues. For a cut-off value of 95 for instance, theestimated sensitivity and specificity for the adhesiontest F4ac equal, is respectively 23% and 84%. Basedon the experiments of Cox and Houvenaghel (1993), acut-off value of 5 bacteria was chosen. With this cut-off value the estimated sensitivity and specificity forthe adhesion test for both F4ab and F4ac equalrespectively 100% and 24%.The estimated variance between measurements inthe same pig equals 10.2 and 13.0 for F4ab and F4ac,respectively. The estimated pig variance component(the additional variability due to pig) equals 22.2 and43.8, much larger than the within pig variance. Thisvariance is due to the fact that some pigs do not haveany expression (due to the lack of the gene) and thatthe expression of the F4 receptor genes varies whenthe gene is present (Bijlsma and Bouw, 1987). Anotherfactor which could explain the individual variation isthe age of the pigs. The villus length decreases withaging (Cera et al., 1988).It can not be excluded that there could be somedifference in results of the in vitro villous adhesionassay compared to the adhesion assay used in the studyof Jørgensen et al. (2003). They performed theadhesion assay by incubating single enterocytes withbacteria as described by Edfors-Lilja et al. (1986). Tento 20 epithelial cells were examined for adhesion byinterference contrast microscopy. Their results werescored from 1 to 4, where 1 = no bacteria and4 = bacteria adhering to the whole brush border of allcells. We examined 250 mm villous brush border,which contains a lot more than 20 enterocytes and ourcriteria for a receptor negative animal was less than 5adhering bacteria per 250 mm brush border.In a study of Erickson et al. (1994), a receptor fromporcine enterocyte membranes was identified andcharacterized as a mucin-type sialoglycoprotein whichbinds F4ac. However, there was no correlation foundbetween the expression of the mucin-type sialogly-coprotein receptor and adherence of bacteria to thebrush border (Francis et al., 1998). Our results do notexclude mucin 4 as a candidate receptor, but theK. Rasschaert et al. / Veterinary Microbiology 123 (2007) 249–253 253absence of the gene in 30.2% pigs positive in the invitro villous adhesion assay for F4ac and F4ab,indicates that there is at least one other receptor forF4ac/ab E. coli. Grange and Mouricourt (1996)already demonstrated that F4ab can bind to a receptorof the transferrin family but there should also exist anadditional receptor(s) for F4ac. As a consequence, notonly mucin 4 gene polymorphism but also expressionof these other receptor(s) has (have) to be included inthe screening assay for F4ac/ab receptor negative pigs.AcknowledgementsThis work was supported by the Research Fund ofthe Ghent University and the ‘‘FWO-Flanders’’.ReferencesBijlsma, I.G., Bouw, J., 1987. Inheritance of K88-mediated adhesionof Escherichia coli to jejunal brush borders in pigs: a geneticanalysis. Vet. Res. Commun. 11, 509–518.Cera, K., Mahan, D., Cross, R., Reinhart, G., Whitmover, R., 1988.Effect of age, weaning and postweaning diet on small intestinalgrowth and jejunal morphology in young swine. J. Anim. Sci. 66,574–584.Cox, E., Houvenaghel, A., 1993. Comparison of the in vitroadhesion of K88, K99, F41 and P987 positive Escherichiacoli to intestinal villi of 4- to 5-week-old pigs. Vet. Microbiol.34, 7–18.Edfors-Lilja, I., Petersson, H., Gahne, B., 1986. Performance of pigswith or without the intestinal receptor for Escherichia coli K88.Anim. Prod. 42, 381–387.Erickson, A.K., Baker, D.R., Bosworth, B.T., Casey, T.A., Benfield,D.A., Francis, D.H., 1994. Characterization of porcine intestinalreceptors for the K88ac fimbrial adhesin of Escherichia coli asmucin-type sialoglycoproteins. Infect. Immun. 62 (12), 5404–5410.Francis, D.H., Grange, P.A., Zeman, D.H., Baker, D.R., Sun, R.,Erickson, A.K., 1998. Expression of mucin-type glycoproteinK88 receptors strongly correlates with piglet susceptibility toK88+ enterotoxigenix Escherichia coli, but adhesion of thisbacterium to brush borders does not. Infect. Immun. 66 (9),4050–4055.Gibbons, R.A., Sellwood, R., Burrows, M., Hunter, P.A., 1977.Inheritance of resistance to neonatal E. coli diarrhea in thepig: examination of the genetic system. Theor. Appl. Genet.51, 65–70.Girardeau, J.P., 1980. A new in vitro technique for attachment tointestinal villi using enteropathogenic Escherichia coli. Ann.Microbiol. 131 (B), 31–37.Grange, P.A., Mouricourt, M.A., 1996. Transferrin associated withthe porcine intestinal mucosa is a receptor specific for K88abfimbriae of Escherichia coli. Infec. Immun. 64, 606–610.Gue´rin, G., Duval-Iflah, Y., Bonneau, M., Bertaud, M., Guillaume,P., Ollivier, L., 1993. Evidence for linkage between K88ab,K88ac intestinal receptors to Escherichia coli and transferrinloci in pigs. Anim. Genet. 24, 393–396.Gyles, C.L., 1994. Escherichia coli enterotoxins. In: Gyles, C.L.(Ed.), Escherichia coli in domestic animals and humans. CABInternational, Wallingford, Oxon, UK, pp. 337–364.Jørgensen, C.B., Cirera, S., Anderson, S.I., Archibald, A.L., Raud-sepp, T., Chowdhary, B., Edfors-Lilja, I., Anderson, L., Fred-holm, M., 2003. Linkage and comparative mapping of the locuscontrolling susceptibility towards E. coli F4ab/ac diarrhea inpigs. Cytogenet. Genome Res. 102, 157–163.Jørgensen, C.B., Cirera, S., Archibald, A.L., Anderson, L., Fred-holm, M., Edfors-Lilja, I. 2004. Porcine polymorphisms andmethods for detecting them. International application publishedunder the patent cooperation treaty (PCT). PCT/DK2003/000807 or WO2004/048606-A2.Peelman, L.J., 1999. Genetic investigation of the resistance mechan-isms of the pig against diarrhea caused by E. coli. Verh. K. Acad.Geneeskd. Belg. 61, 489–515.Sellwood, R., Gibbons, R.A., Jones, G.W., Rutter, J.M., 1975.Adhesion of enteropathogenic Escherichia coli to pig intestinalbrush borders: the existence of two pig phenotypes. J. Med.Microbiol. 8, 405–411.Van den Broeck, W., Cox, E., Goddeeris, B.M., 1999. Receptor-specific binding of purified F4 to isolated villi. Vet. Microbiol.68, 255–263.

Screening of pigs resistant to F4 enterotoxigenic Escherichia coli (ETEC) infection

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Screening of pigs resistant to F4 enterotoxigenic Escherichia coli (ETEC) infection

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