Methodology and application of Escherichia coli F4 and F18 … · 2019. 6. 13. · REVIEW Open...

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Methodology and application of Escherichiacoli F4 and F18 encoding infection modelsin post-weaning pigsDiana Luise1, Charlotte Lauridsen2, Paolo Bosi1 and Paolo Trevisi1*

Abstract

The enterotoxigenic Escherichia coli (ETEC) expressing F4 and F18 fimbriae are the two main pathogens associatedwith post-weaning diarrhea (PWD) in piglets. The growing global concern regarding antimicrobial resistance (AMR)has encouraged research into the development of nutritional and feeding strategies as well as vaccinationprotocols in order to counteract the PWD due to ETEC. A valid approach to researching effective strategies is toimplement piglet in vivo challenge models with ETEC infection. Thus, the proper application and standardization ofETEC F4 and F18 challenge models represent an urgent priority. The current review provides an overview regardingthe current piglet ETEC F4 and F18 challenge models; it highlights the key points for setting the challenge protocolsand the most important indicators which should be included in research studies to verify the effectiveness of the ETECchallenge.Based on the current review, it is recommended that the setting of the model correctly assesses the choiceand preconditioning of pigs, and the timing and dosage of the ETEC inoculation. Furthermore, the evaluationof the ETEC challenge response should include both clinical parameters (such as the occurrence of diarrhea,rectal temperature and bacterial fecal shedding) and biomarkers for the specific expression of ETEC F4/F18(such as antibody production, specific F4/F18 immunoglobulins (Igs), ETEC F4/F18 fecal enumeration andanalysis of the F4/F18 receptors expression in the intestinal brush borders). On the basis of the review, thepiglets’ response upon F4 or F18 inoculation differed in terms of the timing and intensity of the diarrheadevelopment, on ETEC fecal shedding and in the piglets’ immunological antibody response. This informationwas considered to be relevant to correctly define the experimental protocol, the data recording and thesample collections. Appropriate challenge settings and evaluation of the response parameters will allow futureresearch studies to comply with the replacement, reduction and refinement (3R) approach, and to be able toevaluate the efficiency of a given feeding, nutritional or vaccination intervention in order to combat ETEC infection.

Keywords: Biomarkers, Challenge, ETEC, Piglet, Post-weaning

IntroductionPost-weaning diarrhea (PWD) appears primarily duringthe first 2 weeks post-weaning of the piglet. Accordingto the literature, the most diffuse etiological agents re-sponsible for PWD in piglets are enterotoxigenic Escher-ichia coli (ETEC) displaying the fimbriae F4 and F18. Tocontrol the risk related to the occurrence of PWD, theimproper use of antibiotic treatment during the first 2

weeks post-weaning is prevalent in pig production. Asan alternative to treatment with antimicrobials, the ad-ministration of the supranutritional level of zinc oxide(ZnO) at 2500–3000 ppm is a common strategy; how-ever, this strategy has been banned by the EuropeanUnion (EU) Commission beginning in 2022 [1]. The in-creased awareness of the use of antibiotics and ZnO isdue to the growing risk of the occurrence of antimicro-bial resistance (AMR) and of their environmental im-pact. In Europe, a recent limitation regarding the use ofantibiotics, even for therapeutic purposes (e.g., colistin),has arrived. Hence, there is an increased and emergent

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: [email protected] of Agricultural and Food Sciences (DISTAL), Alma MaterStudiorum - University of Bologna, Bologna, ItalyFull list of author information is available at the end of the article

Luise et al. Journal of Animal Science and Biotechnology (2019) 10:53 https://doi.org/10.1186/s40104-019-0352-7

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interest in developing new strategies to limit the occur-rence of PWD in pig production, and scientists, veteri-narians, and nutritionists are trying to identify solutionsfor preventing and treating PWD. However, this is amajor challenge and, according to the authors’ know-ledge, no ‘silver bullet’ has yet been identified to copewith PWD. Previous reviews have described nutritionaland feeding strategies, such as supplementation with or-ganic and inorganic acids [2], essential oils and herbs[3], and some types of probiotics, prebiotics and symbi-otics [4], different dosages of essential amino acids [5]and nucleotides [6, 7], or the potential use of bacterio-phages [8] to prevent and counteract PWD. In order toresearch effective strategies with the potential of coun-teracting PWD, a valid approach is to implement in vivochallenge models with ETEC infection.The most diffuse in vivo challenge models are based on

lipopolysaccharide (LPS); ETEC or ETEC twinned withcircovirus. LPS is the outer surface of all Gram-negativebacteria; it causes acute immune stimulation by means ofthe activation of several signalling pathways, (e.g., TLR4and CD14) resulting in a cascade of syntheses of cyto-kines, miming many aspects of the inflammatory processof pathogens [9, 10]. However, the in vivo challenge modelwith LPS poses some concerns including 1) the develop-ment of endotoxin tolerance by the host, defined as re-duced responsiveness to the LPS [11] which mayconfound the results of the in vivo trial and 2) the limita-tion of studying the direct effects of feeding additives andvaccines during the challenge (e.g., competitive exclusion,toxin binding, etc.) which is mainly important in studiesaimed at testing the ability of some additives in counter-acting PWD. Although the ETEC challenge model hasbeen widely used in several studies testing additives andvaccines to counteract PWD [12–17], the prevalence ofpigs showing signs of infection could be low and highlyvariable among studies. Thus, there is a demand foroptimization of the methodology and standardization ofthe control points in order to assure the appropriate appli-cation of the ETEC challenge model in post-weaning pigs.Therefore, this review provides an overview and evalu-ation regarding 1) the current piglet ETEC F4ac and F18infection models and 2) the key clinical parameters andbiomarkers of the disease which should be included in theexperimental research. An additional aim of the presentreview was to improve the effectiveness of the protocolsbased on the challenge model with ETEC in order to com-ply with the Replacement, Reduction and Refinement(3Rs) principles, especially the Reduction and Refinementapproaches as recently defined by Clark [18].

Literature searchA literature search was conducted using PubMed, Goo-gle Scholar, Web of Science, and Scopus. The main aim

of the literature research was the evaluation of ETEC F4and F18 challenge studies in weaned piglets. Researcharticles in scientific journals, which were published from1997 to January 2019 were primarily considered for dataextraction for both the ETEC F4 and ETEC F18 chal-lenge models. The following search terms in differentcombinations were applied to identify acceptable articles:Escherichia coli; ETEC F4 (and ETEC K88, according tothe previous classification), ETEC F18 (and ETEC F107,2134P and 8813, according to the previous classifica-tion); fecal score; post-weaning diarrhea and pig/por-cine/piglet. Furthermore, published research studiesbased only on in vitro experiments were excluded fromthe studies considered.

F4 and F18 ETEC and their putative receptors inpigletsEnterotoxigenic Escherichia coli strains are characterizedby two types of virulence factors: 1) adhesins whichallow their binding to and the colonization of the intes-tinal epithelium and 2) enterotoxins causing fluid secre-tion. The adhesins are expressed in the ETEC fimbriae,and differ between ETEC F4 and ETEC F18. Detailed in-formation regarding fimbrial structure has been reportedby Dubreuil et al. [19]. In addition, a non-fimbrial adhe-sin referred to as the adhesin involved in diffuse adher-ence (AIDA) has been recognized in ETEC strainsisolated from weaned piglets with PWD [20, 21]; how-ever, its role in PWD has still to be elucidated [22].Once the ETEC have adhered and colonized the small

intestine, they can produce enterotoxin(s) leading to diar-rhea. Both ETEC F4 and F18 are recognized as producingtwo classes of enterotoxins, heat labile (LT) enterotoxinsand heat stable (STa, STb and enteroaggregativeheat-stable toxin 1 [EAST1]) enterotoxins causing electro-lyte and net fluid losses [23, 24].Currently, three serological variances of F4 have been

described, namely F4ab, F4ac and F4ad, and of these, theF4ac variant has been recognized as the most prevalentin piglets [24]. Despite the differences in the antigenicclassification of F4 serological variances, a commonmajor fimbria sub-unit FaeG has been recognized as theF4 adhesin [25].Many putative receptors have been identified for ETEC

F4 adhesion showing various chemical natures and variousmolecular weights, as has been reported in other reviews[19, 26, 27]. Focusing on F4ac, one of the widely acceptedputative receptors is constituted by two intestinalmucin-type sialoglycoproteins (IMTGP-1 and IMTGP-2)[28] linked by galactose [29]. However, these intestinalmucin-type glycoproteins have not been recognized as be-ing responsible for transcytosis and for inducing a sufficientimmune response. Thus, aminopeptidase N (APN) hasbeen recognized as an F4 receptor (F4R) involved in the

Luise et al. Journal of Animal Science and Biotechnology (2019) 10:53 Page 2 of 20

endocytosis of ETEC F4, even if it is not restricted to F4but is also known as a receptor for some coronaviruses [30,31]. Furthermore, a number of additional putative receptorswith a glycosphingolipid nature, such as lactosylceramide,gangliotriaosylceramide, gangliotetraosylceramide, globo-triaosylceramide, lactotetraosylceramide, and lactotetraosyl-ceramide have been proposed [29, 32, 33].Regarding ETEC F18, to date, two antigenic variants

have been identified: F18ab (previously known as F107)and F18ac (previously known as 2134P and 8813) [34].The majority of ETEC F18 strains are able to produceheat-stable enterotoxins including STa and STb [35]while the ability to produce the Shiga toxin has beenmore associated with F18ab [25, 36, 37]. In addition,ETEC F18ac and F18ab differ regarding their adhesioncapacity; the ETEC F18ab displayed a weaker capacity toadhere both in vivo to ligated intestinal loops of weanedpiglets and in vitro as compared to ETEC F18ac [37, 38].The F18 ETEC adhere to the glycoproteins on themicrovilli of the small intestine by means of their minorfimbrial subunit FedF [38, 39]. To date, a putative por-cine enterocyte receptor for ETEC F18 (F18R) has beenrecognized to be the H-2 histo-blood group antigen(HBGAs) or its derivative A-2 HBGAs [40]. A detaileddescription of the ETEC F4 and F18 pathogenesis hasbeen reviewed by Nagy et al. and Peterson et al. [41, 42].

Setting of the challenge modelUp to now, several protocols have been published to im-plement the ETEC challenge model in piglets. In fact,the ETEC challenge can be carried out orally by gastricgavage or following a natural ETEC propagation by in-fecting a few animals in the group. The differences inthe choice and preconditioning of the piglets beforeETEC inoculation have been identified and should beevaluated. In addition, the timing and dosage of theETEC inoculation, as well as the opportunity of supply-ing repeated dosages of ETEC, should be taken intoaccount.

Animal selectionOf the studies reviewed, only a few described thepre-existing sanitary conditions of the farm from whichthe piglets were selected. In the study of Kyriakis et al.[43], the animals were chosen from a farm with poor en-vironmental and management conditions, and in whichthe piglets already exhibited the ETEC F4 infection.Other studies, including Trevisi et al. [44] and Spitzer etal. [45], took piglets from farms in which previous casesof ETEC infection had occurred in order to increase theprobability of having ETEC-susceptible animals. Re-sponses to ETEC F4 and F18 infection showed high indi-vidual animal variability, which can partially beexplained by the animals’ genetic mutations associated

with the expression of specific receptors on the intestinalepithelium. In order to reduce this variability, the choiceof animal can benefit from specific genetic markers asso-ciated with ETEC susceptibility, which could be imple-mented starting from the genotyping of the sows and/orfollowed by piglet genotyping as described in studiesconducted primarily at University experimental facilities[15, 44–47]. A wide range of genetic markers has beenassociated with piglet resistance to ETEC F4 and F18utilizing association studies.For ETEC F4, single nucleotide polymorphisms

(SNPs) located on Mucin4 (MUC4) [48], on Mucin13 (MUC13) [49, 50], Mucin 20 (MUC20) [51], thetransferrin receptor (TFRC) [52], tyrosine kinasenon-receptor 2 (ACK1) [53], UDP-GlcNAc:betaGalbeta-1,3-N-acetylglucosaminyltransferase 5 (B3GNT5)[52] genes have been proposed as genetic markers forpig ETEC resistance/susceptibility. Goetstouwer et al.[54] have recently proposed new SNPs located on thecandidate region (chr13: 144810100-144993222) asnew determinates for ETEC F4 susceptibility. Theproposed SNPs are located on a non-coding regionand may correspond to a porcine orphan gene or atrans-acting element, which make it difficult to applythose markers as screening for the in vivo challengeexperiments. All the above-mentioned markers areconsidered to be candidate markers but none of themhas yet been confirmed as the univocal causative genefor F4 ETEC susceptibility, although all these markersmap in the same q41 region of chromosome 13. Thepolymorphism located in the MUC4 gene appears tobe that most studied. Genetic population studiesbased on MUC4 markers have shown that geneticsusceptibility to ETEC F4 varies according to thebreed. A higher prevalence of MUC4 susceptible pigshas been observed in commercial breeds, such as theLarge White, Landrace and Ukrainian breed pig lineswhile a lower frequency for the susceptible allele hasbeen reported in local breeds [55, 56]. Genetically suscep-tible pigs showed higher diarrhea incidence and greaternumbers of fecal ETEC shedding than genetically resistantanimals; conversely, the phenotypic expression of F4 re-ceptors in the intestinal brush borders displayed a largevariability [57]. Based on the in vitro adhesion test, 30.2%of the MUC4 genetically resistant animals showed specificreceptors for F4ac and F4ab adhesion on the intestinal villi[58]. Thus, it is believed that F4 susceptibility involvesgene epistasis. Furthermore, this could also be due to thelimitation of the MUC4 genotype as the causative gene forETEC F4 susceptibility. However, since genetically F4 sus-ceptible animals (MUC4GG and MUC4CG) showed acomplete phenotypic correspondence to their responseafter ETEC F4 inoculation, the choice of susceptible ani-mals based on pig genotyping may contribute to reducing


individual variability in response to ETEC F4 inoculation[57]. To overcome this lack of association between MUC4genotypes and ETEC F4 susceptibility, the new markersproposed by Goetstouwers et al. [54] should be studiedmore in depth. In fact, since Goetstouwers’ markers mapon a non-coding region, no protocols in addition to anIllumina chip or a next-generation sequencing (NGS)technique are available for pig genotyping. Therefore, add-itional studies are necessary for developing and standard-izing a fast and cheap laboratory method for piggenotyping of the markers detected by Goetstouwers [54]in order to improve pig selection for an ETEC F4 chal-lenge model.Regarding pig resistance to ETEC F18 infection, two

main SNPs localized on alpha (1,2)-fucosyltransferase(FUT1) [59–61] and bactericidal/permeability-increasingprotein (BPI) [62] genes, respectively, have been pro-posed. Greater consensus has been attained for the SNPlocated on FUT1. Data regarding the distribution ofthese genetic markers in pig populations are still scarce.However, Syrovnev [56] observed a high prevalence ofsusceptible genotypes in Ukrainian meat bread pigs andBao et al. [63] showed that, for the most part, the Durocand Pietrain breeds presented the FUT1 resistant(FUT1AA) genotype while wild boar and other Chinesepig breeds only presented the susceptible genotypes(FUT1AG and FUT1GG). In addition, the authors ob-served less scientific research regarding the study of gen-etic influence for ETEC F18 susceptibility than for ETECF4 as compared to the literature research in the presentpaper. This may be due to the fact that less attentionhas been paid to F18 ETEC infection as compared toETEC F4 infection, except for countries such asDenmark, in which breeding programs already selectedfor F4 pig resistance have resulted in a reduction ofF4-susceptible pigs from the Danish swine population.In the present literature review, it was observed that

few in vivo ETEC infection studies included the selectionof piglets based on genetic markers associated withETEC susceptibility (Table 1).For ETEC F4, a total of fifteen studies were found and,

of these, the most frequently used genetic markers werepresent in the SNP located on MUC4, for which genotyp-ing was applied in ten out of the fifteen studies. Pig geno-typing has been applied for different purposes. In thestudies of Fairbrother et al. [14], Trevisi et al. [12, 33],Sørensen et al. [64] and Sugiharto et al. [65], the pigs weregenotyped for the MUC4 genetic marker in order tochoose the genetically-susceptible pigs to be included inthe trial. With the same purpose, Girard et al. [46]adopted the MUC13 genetic marker while both geneticallysusceptible and resistant pigs were included in the studiesof Nadeau et al. [66] and Sargeant et al. [67] with the pur-pose of investigating the differences in kinetics and

localization of the immune response for the developmentof an effective vaccine. On the other hand, Yang et al. [68],Zhang et al. [69] and Zhou et al. [70] decided to includegenetically resistant animals (MUC4-negative pigs) in anin vivo challenge studies with a specific ETEC F4 hybridexpressing virulence factors STb, LT and Stx2e, attachingand effacing intimin (eae), translocated intimin receptor(tir), escV, and E. coli-secreted protein A (espA). Thesestudies showed that ETEC strains with different virulencecapacities can cause enteritis in MUC4-resistant piglets.Nevertheless, it is important to note that MUC4 has beenindicated as a marker for the ETEC F4ac receptor(F4acR), and that this strain is characterized only by STb,LT and EAST1 enterotoxins [71]; thus it is possible thatdifferent F4 strains can induce infection in more complexmechanisms which are yet to be elucidated.To date, nine studies which included pig choice based on

the genetic marker for ETEC F18 resistance have been re-ported (Table 1). Genetically susceptible piglets (for theFUT1 marker) have been included in studies to determinethe kinetic dynamics of immune responses [72], plasmametabolites and immune response [17] to testimmunization strategies, including vaccines [66, 73, 74], orto test additives to protect against infection [15, 75, 76].Furthermore, three out of nine studies were carried out onnewborn piglets in order to propose the ETEC F18 chal-lenge as a model for humans [75–77]. Although studies re-garding infectious challenge models based on FUT1 arescarce, more recent studies carried out on healthy pigletshave pointed out that the FUT1 genotypes can influencethe intestinal microbial profile [78, 79], the expression ofthe intestinal genes [80], intestinal mucosa protein glycosyl-ation [81], piglet blood metabolomics [78, 79], and pigletgrowth performance [82] under normal healthy conditions.Thus, implementation of the FUT1 marker in future ETECF18 challenge studies would be beneficial in order to reducethe variability due to the genetic effect in the response data.In addition to piglet screening for pathogen suscepti-

bility, the pathogen-specific immunization of piglets andsows should be evaluated. In fact, beyond the passiveimmunity derived from sow’s milk which can affect thepiglet’s responsiveness to ETEC immediately after wean-ing, it has been shown that maternal immunity can per-sist in piglet blood and can induce a systemic immuneresponse in the piglets [83], resulting in a less efficientpiglet response to the ETEC challenge. Therefore, instudies where feeding strategies with the aim of con-tracting the ETEC infection, selecting piglets from sowsnot specifically immunized for ETEC and not infectedwith the pathogen previously has been recommended.For studies where vaccine strategies are tested, the pas-sage of maternal immunization should be considered fora correct interpretation of the results, as suggested byNguyen et al. [83].


Table

1Listof

ETEC

F4andF18challeng

etrialsinclud

inganim

alchoicesforsuscep

tibility,the

irrelativemod

elsettings

andtheob

served

infectionindicatorsinclud

ed

Na

Gen

eticsvariantsb

No.of

pigs

per

grou

pWeaning

Con

ditio

ning

ETEC

cChalleng

edo

seChalleng

etim

ingd

Con

trol

grou

peIndicatorsforchalleng

eeffectiven

ess

Endof

thetrial

Reff

1MUC4

−/−

+/−

and

+/+

8d2

8No

F4ac

109CFU

/mL

3dp

wNA

NA

7dp

i[67]

2MUC4

−/−

8d2

1No

F4ac

10mLof

ETEC

cultu

re10

9CFU

/mL

8dp

wNeg

Diarrhe

a;RT

i7dp

i[70]

3MUC4

−/−

6d2

1No

F4ac

10mLof

ETEC

cultu

re10

9CFU

/mL

8dp

wNeg

Diarrhe

a;RT

i7dp

i[69]

4MUC4

−/−

8d2

1No

F4ac

1×10

10CFU

/mL

8dp

wNeg

Abu

ndance

ofE.coliin

feces

7dp

i[68]

5MUC4

+/+

10d2

4No

F4ac

5mLof

suspen

sion

containing

108CFU

/mL

7dp

wNA

Diarrhe

a;fecalETECplatecoun

ts;

anti-F4

IgAandIgM

inbloo

d14

dpi

[12]

6MUC4

+/+

5d2

4No

F4ac

5mLof

suspen

sion

containing

108CFU

/mL

7dp

wNA

Diarrhe

a;In

vitroadhe

sion

test

24hpi

[44]

7MUC4

+/+

10or

20d1

7–18

Yes

F4ac

1.5×10

9CFU

/mLor

1.7×10

9CFU

/mLor

9.8×10

8CFU

/mL

3or

7or

21dp

wNA

Diarrhe

a;gu

tconten

tconsistency;

ETEC

F4fecalshe

dding;

serum

and

intestinalanti-F4

IgM,IgA

andIgG

3or

5dp

i[14]

8MUC4+/+

8d4

2Yes

F4ac

1×10

8CFU

in20

mLNaC

lFrom

1to

3dp

wNeg

Diarroe


ts,

IgAandIgM

inbloo

d10

dpw

[64]

9MUC4+/+

6d2

8Yes

F4ac

1×10

8CFU

in20

mLNaC

lFrom

1to

2Neg

Diarroe


ts12

dpw

[65]

10vanHaerin

gen

labo

ratory

MUC4+/+.

gExp1

=3;

gExp2

=6

d28

Yes

F4ac

1×10

9CFU

/mLin

5mL

of9%

NaC

l+10

mLof

10%

NaH

CO3

2dp

wExp1

:Neg

;Exp2

:NA

gExp1

andExp2

:Diarrhe

aandfecal

DM;fecalhaem

olicE.coli;

19dp

i[91]

11vanHaerin

gen

labo

ratory

MUC4+/+

8–10

d30–32

Yes

F4ac

5×10

7CFU

in20

mLNaC

lFrom

1to

2dp

wNeg

Diarroe


ts10

dpw

[15]

12MUC13+/+

gExp1

=16;

gExp2

=18

d24

No

F4ac

5mLof

108CFU

/mL

4dp

wNeg

gExp1

:Diarrhe

a;Exp2

:Diarrhe

a;fecal

ETEC

quantification

13dp

i[46]

13MUC4

+/+;FUT1

+/+

F4=20;

F18=18

d28

Yes

F4and

F18ac

F18ac:10

11CFU

/mL;F4

1011CFU

/mLfor2

days

7dp

wNeg

Diarrhe

al;ETECF18andF4

fecal

shed

ding

;F18

andF4

specific

antib

odiesin

bloo

d;in

vitro

adhe

sion

test

20dp

i[72]

14Exp1

:FUT1

+/+

and

MUC4

−/−

orMUC4

+/+;Exp2:FU

T1+/+

andMUC4

+/+

gExp1

=12;

gExp2

=10

d16–18

No

gExp1

:F18;

gExp2

:F4

Exp1

=1×10

10CFU

/mL

or5×10

10CFU

/mL;

Exp2

=1.3×10

9CFU

/mL

7or

21dpo

stvaccination

Neg

Diarrhe

a;ETEC

F18andF4

fecal

shed

ding

;Serum

anti-F4

andanti-

F18IgM

andIgA;

Exp1

=7

dpi;

Exp2

=4

dpi

[66]

15MUC4

+/+

and

FUT1

+/+

6NA

No

F18ab

10mLof

1011CFU

/mL

62dp

wNA

FecalETECF18;serum

IgA;invitro

adhe

sion

test

7dp

i[73]

16FU

T1+/+

6gExp1

:d25;

gExp2

:d30

Yes

gExp1

:F18ab;

gExp2

:F18ac

gExp1

:5mLof

6.7log

CFU

;gExp2

:5mlo

f6.7

logCFU

Exp1

:11dp

w;Exp2:

13dp

w+30

dpw

the2°

challeng

e

NA

ETEC

F18fecalcou

nt;serum

F18

IgA

8dp

i[113]


Table

1Listof

ETEC

F4andF18challeng

etrialsinclud

inganim

alchoicesforsuscep

tibility,the

irrelativemod

elsettings

andtheob

served

infectionindicatorsinclud

ed(Con

tinued)

Na

Gen

eticsvariantsb

No.of

pigs

per

grou

pWeaning

Con

ditio

ning

ETEC

cChalleng

edo

seChalleng

etim

ingd

Con

trol

grou

peIndicatorsforchalleng

eeffectiven

ess

Endof

thetrial

Reff

17FU

T1+/+

gExp1

=4;

gExp2

=3

d28

Yes

F18ab

10mLof

1011CFU/mL

atweaning

NA

Diarrhe

a;F18-specificantib

odiesin

bloo

d;in

vitroadhe

sion

test

8dp

i[47]

18FU

T1+/+

9Not

pertinen

thNo

F18

1mLof

7.5×10

10

CFU

/mL

from

days

1–8of

life

Neg

Diarrhe

a;E.coliabou

ndance

inintestinalconten

t8dp

i[75]

19FU

T1+/+

10Not

pertinen

thNo

F18

1mLof

1010CFU

/mL

from

days

1–4of

life

Neg

Diarrhe

a5dp

i[76]

20FU

T1+/+

gExp1

=6;

gExp2

=5

gExp1

=d2

8;gExp2

=d2

1F18

10mLof

1011CFU/mL

gExp1

:22dp

w;

gExp2

:7dp

wExp1

:Neg

;Exp2

:NA

gExp1

andExp2:Fecalexcretio

nof

ETEC

F18;F18-

andFedF-spe

cific

IgM,IgA

andIgGrespon

sein

bloo

dandsaliva;in

vitroadhe

sion

test

Exp1

:18

dpi;

Exp2

:21

dpi

[74]

21FU

T1+/+

18d2

8–34

No

F18

50mLof

3×10

8

CFU

/mL

From

3to

6dp

wNA

Diarrh

ea;E.coliand

Hem

olytic

bacteriacoun

ton

feces;FecalD

M;

plasmaIgAandIgG

Not

repo

rted

[17]

a NStud

ynu

mbe

rbGen

eticsvaria

nts:MUC4

:Mucine4

;MUC1

3:Mucine1

3;FU

T1:Fucosyltran

sferase1.

+/+

stan

dsforthege

notype

sassociated

with

thesuscep

tibility

totherespectiv

eETEC

;−/−:the

geno

type

sassociated

with

theresistan

cec ETECEn

terotoxige

nicEscherichiacoli

dCha

lleng

etim

ing:

itwas

repo

rted

asda

yspo

st-w

eaning

(dpw

)e Con

trol

grou

p:Neg

Neg

ativecontrolg

roup

(not

infected

),Po

sPo

sitiv

econtrolg

roup

(group

treatedwith

antib

iotic)

f Ref.R

eferen

cegExpExpe

rimen

thNot

pertinen

t:pe

rformed

inne

wbo

rncesarean

-delivered

pigs

i RTRe

ctal

tempe

rature

Add

ition

alab

breviatio

ns:D

day,CF

UColon

yform

ingun

its,N

ANot

available,

dpiD

ayspo

st-in

oculum


Animal pre-conditioningPreconditioning procedures should be carried out tocontain the within variability of the piglets’ response tothe ETEC challenge on the basis of their physiologicalstatus before the infection. Among preconditioning pro-cedures, pigs can initially be treated with antibiotics, in-cluding colistin (50/60 mg per pig) [45, 84–86] orflorfenicol (2 mL per pig) [87], in order to keep the ani-mals in a healthy condition before ETEC inoculation orto contrast the effects of the weaning transition. How-ever, this practice poses some risks; in fact, the pro-longed administration of antibiotics can reduce the gutmicrobial variability, compromise the gut eubiosis andimpair animal health [88]. Therefore, the potential anti-biotic administration should usually be restricted tonarrow-spectrum antibiotics and only for the first 3–4 dpost-weaning [13, 89].Furthermore, an additional practice for increasing and

standardizing the piglets’ response to ETEC inoculationconsists of having the animals fast for 3 h before infectionand then subsequently administering 62mL of a 1.4%NaHCO3-solution in order to neutralize the gastric pHbefore ETEC inoculation [90]. This procedure has beenapplied mainly in studies aiming to test immunizationstrategies [72, 73, 91].

Control groupsOverall, twenty-six out of forty-eight studies included anadditional negative control group (Tables 1 and 2). In-cluding a negative control group is recommended for invivo experiments and could be mandatory in experi-ments testing drugs [92]. This could represent a criticalaspect in the case that insufficient parameters of proveninfection are included in the study. However, if a goodhealth status of piglets is guaranteed before the ETECinoculation and a positive control group is included (i.e.an antibiotic group), the negative control group could beredundant [93]. On the other hand, if it is hypothesizedthat a given feed additive or a nutritional treatment is in-fluencing the progression of the PWD via immuno-logical mechanisms it is recommended to include anon-challenged group with the same dietary treatment.

Timing of the inoculumThe timing of ETEC inoculation is an important pointto consider for a successful pig challenge model.The expression of F4R on the brush border membrane

of the small intestine has been reported to be equallypresent at 1 week, 5 weeks and 6months of age [94].While contradictory results have been reported for theexpression of F4R in the mucosa of the small intestine,Willemsen and de Graaf [94] observed no difference in7-day-old and 35-day-old piglets and only rare detectionof F4R in 6-month-old pigs. Conway et al. [95] reported

an increase in F4R expression in from 7-day-old pig-lets up to 35-day-old pigs. In the first weeks of life,the increase in F4R expression in the mucosa accord-ing to increased age has also been proposed as one ofthe mechanisms which favor ETEC F4 infection inpiglets [95].Scarce information is available regarding the age-

depended expression of F18R. The in vitro adhesion teston porcine intestinal villi showed the absence of F18R atbirth in genetically-susceptible piglets; it then increasedin 3-week-old piglets, and subsequently higher expres-sion appeared post-weaning and was maintained until 23weeks of age [40]. However, the results reported byNadeau et al. [66] showed an increase in the specific im-mune response (F18-specific IgA) and in diarrhea sever-ity in 18-day-old pigs, suggesting that F18R was alreadyexpressed at this age. Furthermore, a positive responseto ETEC F18 inoculation has been observed in 0- to7-day-old cesarean-delivered piglets, supporting the the-ory that F18R could be present in the early phase of life[77]. Additional experiments are required to draw a con-clusion regarding the age-dependent presence of F18Rsince the divergent results obtained until now are diffi-cult to compare due to differences in detecting the F18Ras well as to differences in the experimental conditions.Overall, the age-dependent expression of F4 and F18

receptors in the small intestine could contribute toexplaining why ETEC F4 infection mainly occurs duringthe neonatal period and at weaning while ETEC F18 in-fection mainly occurs together with weaning and later inthe piglet’s life during the growing period.Furthermore, the multifactorial stress of weaning

followed by a drop in passive immunity increases therisk of developing intestinal dysbiosis, and subsequentcolibacillosis due to ETEC [96–98].To take advantage of the stressful situation and

intestinal dysbiosis, which characterize weaning, some au-thors have carried out the ETEC F4 or F18-inoculation onthe day of weaning [16, 99] or one-day post-weaning [89,100–102]. However, it should be considered that the pas-sive immunity derived from sow milk immunoglobulinscan influence the piglet’s response to the pathogen, caus-ing reduced infection effectiveness. Therefore, the major-ity of studies have carried out the first ETEC challenge atfrom 3 or 4 d post-weaning [45, 46, 67, 86, 103] to 1-weekpost-weaning [12, 44, 104–106] due to the considerationthat, during this time period, passive immunity decreased,and the piglets were still affected by the critical issuesresulting from weaning. However, the effectiveness of theETEC challenge probably depends on the weaning ageand on the weight of the piglet. In studies in which theETEC F4 inoculation was carried out 14 d post-weaning(dpw), no problem of passive immunity can be expected[107, 108]; however, the piglets could have acquired a


Table

2Listof

ETEC

F4andF18challeng

etrialsrelativeto

themod

elsettingandtheinfectionindicatorsob

served

Na

No.of

pigs

per

grou

pWeaning

Con

ditio

ning

ETEC

bChalleng

edo

seChalleng

etim

ingc

Con

trol

grou

pdIndicatorsforchalleng

eeffectiven

ess

Endof

thetrial

Ref.e

164

d28

No

F4ac

infarm

NA

Alreadypresen

tat

weaning

NA

Fecalscore;p

ost-mortem

exam

inations

28dp

w[43]

224

d25

Yes

F4ac

10mLof

8.1log 1

0CFU

/mL

4dp

wNA

Fecalscore;cou

ntingof

haem

olitic

bacteria;invitroadhesion

test

26dp

i[86]

36

d18

No

F4ac

1×10

9CFU

/mL

1dp

wNeg

.and

pos

Fecalscore;fecalcoliformscoun

t6dp

i[101]

48

d28

No

F4+O149

2×10

10CFU

/mL

From

1to

2dp

wNA

Diarrhe

a,ETEC

shed

ding

infeces

11dp

i[100]

524

d28

No

F4+O149

6,8and10

mLof

3.44

×10

8CFU

/mL

From

5to

7dp

wNA

Diarrhe

a;ETEC

fecalshe

dding

7dp

i[112]

68

d28

No

F43mLof

3×10

10CFU

/mL

7dp

wNeg

.group

Diarrhe

a;ETEC

shed

ding

infeces;Po

st-

mortem

exam

ination

7dp

i[104]

76

d25

No

F4%

mLof

5×10

8CFU

/mL

Atweaning

NA

Diarrhe

a;ETEC

shed

ding

infeces

7dp

i[99]

824

d21

No

F46,8and10

mLof

2.16

×10

8CFU

/mL

From

3to

6dp

wNeg

Diarrh

ea:haemolyticE.colifecalshedd

ing

11dp

i[103]

912

d21

Yes

F4ac

2mLof

5.0×10

9CFU

/mL

From

3to

4dp

wNeg

Gen

eralcond

ition

scores

ofpigs

behaviou

r;diarrhea;RTg;fecalfaeand

est-IIconcen

trations

7dp

i[45]

107

d28

No

F41.5mLof

10×10

10

CFU

/mL

14dp

wNA

Diarrhe

a;E.coliin

feces;bloo

dIgs

28dp

i[107]

1110

d24

No

F41.5mLof

10×10

8CFU

/mL

7dp

wPo

sDiarrhe

a;ETEC

shed

ding

infeces;bloo

dtotalIgA

andF4ac-spe

cific

IgA

14dp

i[13]

125

d21

No

F41mLof

1×10

9CFU

/mL

7dp

wNeg

andPo

sDiarrhe

a8dp

i[105]

136

d21

No

F41mLof

1×10

9CFU

/mL

9dp

wNeg

andpo

sDiarrhe

a12

dpi

[115]

145

d28

No

F41×10

8CFU

/mL

14dp

wNeg

sIgA

inbile

2dp

i[108]

1518

d17

No

F430

mLof

analkalinebroth

1010CFU

/mL

7dp

wNeg

Diarrhe

a7dp

i[106]

163–7

NA

Yes

F4NA

Atweaning

Neg

Diarrhe

a;serum

Igs

11dp

i[16]

175

NA

No

F410

9CFU

/mL

12dp

wNeg

Invitroadhe

sion

test

24hafterinfection

[102]

188(28in

COf )

d21

Yes

F4ac

1.5mLsuspen

sion

of10

10

CFU

/mL

5dp

wNeg

Diarrhe

a;bloo

dspecific-F4

IgA;Invitro

adhe

sion

test;ETECplatecoun

tson

intestinalconten

t

50%

on4dp

i;50%

on18

dpi

[116]

1924

d21

No

F41.5mLsuspen

sion

of10

10

CFU

/mL

7dp

wNA

Diarrhe

a;fecalE.coliand

ETEC

coun

t;bloo

dspecific-F4

IgA;Invitroadhe

sion

test

7dp

i[140]

2020

d21

No

F41.5mLsuspen

sion

of10

10

CFU

/mL

2dp

wNA

Diarrhe

a;fecalE.coliand

ETEC

coun

t;bloo

dspecific-F4

IgA;Invitroadhe

sion

test

6dp

i[128]

2112

d21

No

F41.5mLsuspen

sion

of10

104dp

wPo

sDiarrhe

a;ETEC

F4coun

tin

fecesand

11dp

i[89]


Table

2Listof

ETEC

F4andF18challeng

etrialsrelativeto

themod

elsettingandtheinfectionindicatorsob

served

(Con

tinued)

Na

No.of

pigs

per

grou

pWeaning

Con

ditio

ning

ETEC

bChalleng

edo

seChalleng

etim

ingc

Con

trol

grou

pdIndicatorsforchalleng

eeffectiven

ess

Endof

thetrial

Ref.e

CFU

/mL

intestinalconten

t;bloo

dspecific-F4

IgA;

Invitroadhe

sion

test

2212

d21

No

F41.5mLsuspen

sion

of10

10

CFU

/mL

1dp

wNA

Diarrhe

a;ETEC

F4coun

tin

fecesand

intestinalconten

t;bloo

dandsaliva

specific-F4

IgA;invitroadhesion

test

7dp

i[127]

239

d17

Yes

F46mLof

2×2×10

10CFU/m

L9dp

wNA

Diarrhe

a;E.coliqu

antificationin

colon

7dp

i[110]

2412

d49

No

F420

mLNaC

lwith

108

CFU

/mL

From

2to

4dp

wNeg

FecalD

M10

dpi

[118]

2540

d25

No

F43×10

9CFU

/mL

3dp

wNS

Diarrhe

a;fecalE.colicou

nts;

5dp

ior9dp

i[117]

2612

d20

Yes

F18ab

5mLof

1×10

10CFU

/mL

21dp

wNeg

Diarrhe

a;fecalh

aemolitc

colony;b

lood

IgAandIgG;

30dp

i[84]

2718

(28in

COf )

d28

No

F18ac

1011CFU

/mL

Atweaning

for3d

NA

Diarrhe

a;ETEC

F18qu

antificationin

fecesandintestinalconten

t9dp

i;[114]

a NStud

ynu

mbe

rbETEC

Enterotoxige

nicEscherichiacoli

c Cha

lleng

etim

ing:

itwas

repo

rted

asda

yspo

st-w

eaning

(dpw

)dCon

trol

grou

p:Neg

Neg

ativecontrolg

roup

(not

infected

),Po

sPo

sitiv

econtrolg

roup

(group

treatedwith

antib

iotic)

e Ref.R

eferen

cef COCon

trol

grou

pgRT

Rectal

tempe

rature

Add

ition

alab

breviatio

ns:D

day,CF

UColon

yform

ingun

its,N

ANot

available,

dpiD

ayspo

st-in

oculum


higher immune competence to respond to the infection(Tables 1 and 2) [109]. It is rather difficult to assess whenthe immune system of the piglet is fully developed, andseveral factors beyond weaning age and weight probablyinfluence this process. However, generally speaking, pig-lets are considered immunologically stable by 6–8 weeksof age [109].Furthermore, the timing of the challenge can vary ac-

cording to the objective of the study. The majority of thestudies reviewed had the prophylactic effect of feed addi-tives to counteract PWD as the main objective to inves-tigate. According to this, the given feed additive shouldbe provided some days before the ETEC inoculation,and hence, the timing of the challenge could be approxi-mately 1-week post-weaning. A different objective hasbeen proposed by Cilieborg et al. [75] and Andersen etal. [76] in which 1,2-fucosyllactose and Lactobacillusparacasei or Pediococcus pentosaceus in milk formulaswere tested to counteract the ETEC F18 infection innew-born piglets as a model for human infants.

Inoculation method and dosageEnterotoxigenic Escherichia coli infection is commonlyinduced by the pathogen via oral administration. Lessfrequently, the infection has been induced by an intra-gastric inoculum of the pathogen, for the most part instudies aimed at developing vaccines (for ETEC F4 [55];for ETEC F18 [73, 74]). Although the intragastric gavageallows the inoculum dosage to completely reach thegastrointestinal tract, it represents a painful and stressfulprocedure for the piglets. Therefore, in order tominimize the piglets’ pain and comply with the Refine-ment approach expressed in the 3R strategy [18], an oralinoculum should be preferred.

In ETEC F4 infection studies, the dosage of inoculumadministered to weaned piglets varied, being approxi-mately 108 colony-forming units (CFU), i.e., 1 × 108 CFU[100], 5 mL of 1 × 108 CFU [12], 5 mL of 5 × 108 CFU[99]. Higher dosages, 1.5 mL of 1010 CFU and 6mL of2 × 1010 CFU, have been administered by Trevisi et al.[13] and Molist et al. [110], respectively. Other authors in-duced the infection using repeated administration of thesame dosage of ETEC; e.g., 1 × 108 CFU, for two consecu-tive days [64, 65]. In some studies, increased dosages ofETEC F4 were used, i.e. piglets were challenged with 6, 8and 10mL of 3.44 × 108 CFU/mL on days 5, 6 and 7post-weaning [111]; with 6, 8 and 10mL of 2.16 × 108

CFU/mL for three consecutive days post-weaning [103];with 2mL of 5.0 × 109 CFU/mL twice a day on three con-secutive days post-weaning [45]. Despite the difference inthe dosage used for the ETEC F4 inoculation, the firstdiarrhea signs were reported in all studies at approxi-mately 24 h post-inoculum (Fig. 1). Similarly, newborns (3d of age) challenged with 5mL of 1 × 109 CFU developeddiarrhea within 6 h post-inoculum [112].Regarding the ETEC F18 inoculation, pathogenic dos-

ages varied from 5mL of 108 CFU/mL [113], 5 or 10 mLof 1 × 1010 CFU/mL [66, 84] to a higher dosage of 10mL of a 1011 CFU/mL solution used by Coddens al. [47]and Verdonk et al. [72] in weaned piglets (28 and 35 dold respectively), and Tiels et al. [73] in growing pigs(at 62 d post-weaning) while three consecutive dosagesof 1011 CFU/mL were used by Yokoyama et al. [114] inweaned piglets (28 days old). However, diarrhea has alsobeen induced using a lower dosage of ETEC F18 inocu-lation, i.e. 3 × 108 CFU [17].Overall, it can be noted that for both F4 and F18

ETEC challege protocols, the dosages used are very

Fig. 1 State of the fecal score consistency following enterotoxigenic Escherichia coli (ETEC) F4 inoculation. Data from different studies werereported on a fecal scale from 1 (dry) to 5 (watery). Liu et al. [107]: ETEC F4; Trevisi et al. [12]: ETEC F4 ac; Girard [46]: ETEC F4ac (LT+ and STb+);Lee [104]: ETEC F4; Hedegaard [91]: ETEC F4 (serotype O149:F4). Dpi: days post-inoculum


close to the minimum dosage able to induce infection[14]. Furthermore, although the range of dosage in-oculum did not vary very much among the studies,and the pigs developed diarrhea, a high variability indiarrhea severity and diarrhea incidence have beenobserved (see the section “Diarrhea and related indi-cators”). A large experimental variation in diarrheaoutcome can be due to individual animal variabilityamong the studies i.e. genetic susceptibility andanimal immune competence. In addition, the naturalexposure of E. coli from the sow and/or the environmentmay contribute to variation within an experiment.

Evaluation of the challenge effectivenessA wide range of response indicators has been proposedin ETEC challenge studies, including both clinical andbehavioral parameters. Clinical signs for a completediagnosis have recently been described by Luppi [24]while Jensen et al. [71] and Spitzer et al. [45] proposedscoring the pigs according to their general conditionwith a 1–4 point score where 1 = no impairment ofhealth; 2 =mild impairment: reduced activity, atypicalbehavior, reduced feed intake; 3 =moderate impairment:inactivity, weakness, feed refusal and 4 = severe impair-ment: inappetence, dehydration and reduced bodytemperature. However, these parameters have been criti-cized. In fact, they need to be reported by the sametrained person, they are time-consuming and they arenot widely utilized among the studies; thus, they werenot useful for the present review. Therefore, in this re-view, the most acceptable response indicators, whichallowed determining whether the ETEC challenge wassuccessfully carried out were identified and described.The parameters identified included clinical parameters,such as the occurrence of diarrhea, rectal temperature(RT), and stimulation of immune response or the isola-tion of pathogens in the feces. Among the indicators

described, some were considered pathogen-specific,thereby allowing the proper association of the pig re-sponse to the inoculated ETEC strain, resulting in effect-ive evidence of a successful challenge protocol.

Diarrhea and related indicatorsThe development of the clinical disease symptom (diar-rhea) and the related indices, including its frequency andseverity, are the most accepted response parameters forevaluating ETEC infection. Those diarrhea indicatorscan be assessed using different methods, includingevaluation of fecal consistency scores, fecal dry matter(DM) and days of diarrhea.The most frequently used fecal score classification is

summarized in Table 3. The most frequently used fecalscore classification is based on a continuous scale of 5levels which evaluate fecal consistency where 1 = hardand dry feces; 2 = well-formed firm feces; 3 = formedfeces; 4 = pasty feces and 5 = liquid diarrhea [12, 13, 67]or conversely from 1 to 5 where 1 = watery feces and5 = hard feces [45], and where a consistency score > 3 isdefined as a clinical sign of diarrhea. Scoring can be ex-tended to 7 levels and classified for feces consistencyand color, according to the Bristol Stool Scale where aconsistency score > 3 is defined as a clinical sign of diar-rhea [91] or reduced to 4 levels (1 = normal feces, 2 =soft feces, 3 =mild diarrhea, and 4 = severe diarrhea[104, 111] or to 3 level [115] (Table 3).Overall, one of the most important aspects is the col-

lection time of the fecal consistency data. Fecal score re-cording should start from the day before ETECinoculation in order to check that the health status ofthe animals is good before inoculation, and continuedaily during the first-week post-inoculation and, subse-quently, every second day, preferably until the pigletsrecover.

Table 3 Assessment of the pigs fecal score

Color: intense red indicates the threshold for diarrhea classification and its severity


The majority of the studies which carried out theETEC F4 inoculation during the first week post-weaningreported an impairment of fecal score consistency from24 h’ post-inoculum [12, 45, 104] (Fig. 1) while, in neo-natal piglets, the impairment of the fecal score wasalready observed 3 or 6 h after F4 inoculation [112].Thus, it is very important to record the fecal scoreconsistency in the first 24–36 h after ETEC inoculationin order to identify the exact timing of the appearance ofthe diarrhea. Overall, the peak of the diarrhea (the worstfecal score) has been observed to be from 2 to 4 d afterETEC F4 inoculation up to a week later (Fig. 1).Differences in the timing of the diarrhea occurrence

may be due to individual variability. In fact, piglets witha higher expression of F4Rs on the intestinal brushborder showed an earlier manifestation of diarrhea andthe worst fecal score [13, 71, 116].Data regarding the fecal consistency score could also

be reported as diarrhea incidence defined as the percent-age of piglets with diarrhea on a specific day after ETECinoculation. Differences in diarrhea incidence were ob-served among the studies. Considering the positive con-trol group of the different studies, it could be observedthat ETEC F4 inoculation induced a diarrhea incidencereaching 40–50% at 3 d post-inoculum (dpi) [86], 5 dpi[117] and 7 dpi [12] while it reached 80% in the studiesof Pan et al. [115] at 3 dpi. A reduction in diarrhea inci-dence has been observed within 11 dpi by Pieper et al.[117] and Kiers et al. [86] despite the difference in F4ETEC dosages.Continued monitoring of the fecal consistency score

from the day of inoculation to the end of the trial allowedcalculating the days with diarrhea which reflected the ani-mal recovery.

Fecal DM is a frequently used indicator of porcinediarrhea, and it is inversely correlated with the diar-rhea assessed using fecal scoring, i.e. higher fecal DMwhen there is less diarrhea. It is determined in sam-ples obtained from individual pigs taken daily fromday 1 before the challenge until the end of the chal-lenge [45, 64, 91, 118]. Few studies have reportedfecal DM determination in parallel with the diarrheascore, although fecal DM is not prone to subjectiveevaluation as with fecal scoring. In F4 inoculated pig-lets, fecal DM decreased from 24.7% in pre-challengeconditions to 12.9–20.4% during 1 to 3 dpi. A normalfecal DM was then recovered within 5 dpi [45].Information regarding diarrhea due to F18 ETEC in-

oculation is scarce in comparison with that regarding F4ETEC, and studies have shown a high variability in diar-rhea response despite quite similar inoculation dosages(Fig. 2). The high variability in diarrhea response shownin Fig. 2 could be due to the serological variants of theE.coli used in the various studies. In fact, Coddens et al.[47] used E.coli serotype O139:K12:H1, Rossi et al. [84]used E. coli serotype O138 and Yokoyama et al. [114] E.coli serotype O141. A less severe diarrhea outcome wasobserved by Rossi et al. [84] and Yokoyama et al. [114]as compared to Coddens et al. [47]. The more severediarrhea observed by Coddens [47] could also be due tothe choice of genetically susceptible animals. On thecontrary, Verdonck et al. [74] reported, in piglets genet-ically susceptible to ETEC F18 and treated with the sameETEC dosage and strain used by Coddens, a low diar-rhea response. Measuring the fecal consistency and thefecal DM, Sugiharto et al. [17] observed that 30–40% ofETEC F18-susceptible piglets suffered from diarrhea 3–4 d post-weaning, with the first F18 inoculum provided

Fig. 2 State of the fecal score consistency following the ETEC F18 inoculation. Data from different studies were reported on a fecal scale from 1(dry) to 4 (watery). Coddens et al. [47]: E. coli F18ab-positive, E. coli strain107/86 (serotype O139:K12:H1, F18ab+, SLT-IIv+, resistant at 1 mg/mlstreptomycin; Rossi et al. [84]: E. coli F18ab-positive, (serotype O138, VT2e+); Yokoyama et al. [114]: E. coli F18ac, E. coli strain 8199 (serotypeO141ab: H4: F18ac+: STIa, STII)


to piglets at 1 d post-weaning, i.e. a similar trend in diar-rhea development to the F4 inoculation experiments(Fig. 1). Since the genotype cannot discern the magni-tude of the piglets’ susceptibility, the differences ob-served may be due to the different expression of F18Rson the intestinal brush border. In fact, the comparisonof F18R expression between piglets with susceptible ge-notypes still remains to be studied. Furthermore, differ-ences in the occurrence of diarrhea among the studiescould be due to the F18 strain used and to its virulence.For instance, Yokoyama et al. [114] adopted an ETECF18ac strain while other authors used an ETEC F18abstrain. It is difficult to draw a conclusion regarding thetiming and severity of diarrhea due to ETEC F18 inocu-lation with the data available; thus, additional studies arenecessary to correctly describe the diarrhea manifest-ation as a valid criterion for assessing F18 challengeprotocol.

Rectal temperatureAn additional clinical indicator for pig health status isbody temperature. Body temperature is commonlyassessed using RT which has been considered to be oneof the best indicators of core body temperature [119]. Inchallenge studies, the RT is measured daily from day 1before inoculation until 7 dpi, using an electronic therm-ometer [45, 104]. Pig RT ranges from 39.0–39.5 °Cpre-challenge to > 40.0 °C 6 h’ post-inoculation, and itthen decreases gradually. High variability has been re-ported for the time necessary for the rectal temperatureto return to a physiological level. For an ETEC F4 chal-lenge, the timing can vary from 24 h after inoculation[101] to 2 or 3 dpi [104], or to 5 dpi [45]. However,some concerns have been associated with RT detection.Obtaining RT could be time-consuming and is stressfulfor the animals, especially for sick animals. Furthermore,it may be inaccurate due to the presence of watery fecesin the rectum and the animal’s movements [119, 120];therefore, in the present survey, this measurement wasreported in very few studies.

Bacterial fecal sheddingBacterial shedding has been widely recognized as an in-dicator for evaluating the host responses to infection;however, differences in bacterial species and in the tim-ing of the analyses have been observed. The most accur-ate information is provided by the evaluation of ETECF4 and F18 fecal shedding in the period from before in-oculation to 3–4 dpi. This time period after inoculationis required to allow the ETEC to adhere, colonize andproduce toxins in the small intestine.Differences in the time for ETEC F4 and F18 fecal

excretions post-inoculation have been reported. Thepeak of ETEC F4 excretion following the ETEC F4

inoculation (1011 CFU) is at 2 dpi (5.97 × 108 F4 pergram of feces); a sudden decrease in the ETEC F4fecal count then already occurs at 3–4 dpi [72]. Ver-donck et al. reported a similar level of F4 fecal shed-ding [72] at 3–4 dpi using lower F4 ETEC dosages[12 (108 CFU/mL), 13 (1010 CFU/mL)].For ETEC F18, the peak of fecal excretion occurred 3–

5 dpi (9.9 × 107 F18 per gram of feces); contrary to F4excretion, the amount decreased gradually and it re-solved between 9 and 11 dpi [66, 72, 73, 113]. Hence,the intestinal colonization of ETEC F4 seemed some-what faster than for F18. This could be explained by thedifferent amounts of adhesin in the fimbriae of ETEC F4and F18. The adhesion of F4 fimbriae is mediated by themajor subunit FaeG while, for F18 fimbriae, the adhesinis expressed by the minor subunit FedF, resulting in alower ETEC F18 ability to adhere to the specific recep-tors on intestinal enterocytes, causing a lower immuneresponse and slower pathogen excretion [39, 72, 74]. Inaddition, small differences in fecal shedding between thetwo F18ac and F18ab strains can be observed. In fact,the F18ac strain shows a faster reduction in fecal excre-tion than the F18ab strain [113].Overall, the authors observed that the evaluation of F4

and/or F18 fecal shedding was carried out in only seven-teen out of forty-five studies (Tables 1 and 2). Regret-tably, in the authors’ opinion, this was not adequateconsidering the important information that this analysisobtained. Specific protocols for ETEC F4 and F18 isola-tion from feces and their characterization can be foundin Nadeau et al., Verdonck et al. and Loos et al. [23, 66,72]. Briefly, ETEC F4 and F18 isolation consists of dilut-ing 10 g of feces 10-fold in peptone water and subse-quent anaerobic incubation of the dilutions selected into5% bovine blood agar plates containing 50 μg/mL ofnalidixic acid for 24 h at 37 °C. In addition to the fecalcounting, the ETEC colony should be serotyped in orderto verify the strain [121]. Furthermore, assessing andquantifying the pathogenic enterotoxins may be an evenmore precise estimate for controlling the efficacy of theETEC challenge model since the excreted ETEC toxinsindicate the level of infection. The LT, STa and STb en-terotoxins can be assessed using an enzyme-linked im-munosorbent assay (ELISA), a competitive enzymeimmunoassay (EIA), by immunoblotting using a specificmonoclonal antibody [23] or using a quantitative poly-merase chain reaction (qPCR). Specific primers and con-ditions to detect ETEC virulence genes using PCR canbe found in Byun et al. and Khac et al. [122, 123]. Fur-thermore, the precise detection and quantification of theenterotoxins of the ETEC strains inoculated will permitdefining standard virulence ETEC strains for pig chal-lenge models, resulting in a reduction of strain variabil-ity effects.


Other studies have provided information only on totalE. coli fecal shedding [101, 107, 117] or measuring theCFU at the level of the colon [110]. Since E. coli is con-sidered an ubiquitary bacterium, its total increase cannotbe directly associated with the increase in the pathogenicstrain used for the challenge; therefore, the total increasein E. coli is not considered a precise indicator for claim-ing the success of the challenge protocol.

ImmunoglobulinsImmunoglobulins (Igs) are crucial for defending organ-isms from pathogens and are also recognized as keyplayers for clinical, diagnostic and biotechnological ap-plications. Therefore, Igs have been exploited as themain indicators of ETEC infection and their quantifica-tion in challenge experiments have generally been car-ried out using blood serum and saliva, intestinalmucosal samples or bile. Among Igs, IgG and IgM arepartially ineffective for the mucosal surface while IgAcontributes to the host mucosal defense since it im-proves the organism resistance to bacterial proteolyticenzymes and can bind antigens, preventing pathogenscolonization [124]. For this reason, quantifying thesecretory IgA (SIgA) is recommended and, on infection,its concentration should be higher in mucosal and/or bilesamples of ETEC in infected piglets than in non-infectedpiglets, at least at the peak of the infection [108, 125].However, since slaughter of the experimental piglet is ne-cessary to obtain this information, it is not an option and,therefore, the quantification of plasma or serum IgA iscarried out [17, 73] and, in parallel with hematological pa-rameters, the IgA quantification in plasma or serum al-lows following up the infectious response to the ETEC

challenge as demonstrated by Sugiharto et al. [17] andRossi et al. [84]. In addition to IgA, quantification of bloodIgG and IgM could allow obtaining a more accurate de-scription of animal history regarding previous ETEC infec-tion or regarding immunological competence derivedfrom the mother.In order to obtain the information most targeted to

the response against ETEC F4 and F18, the quantifica-tion of pathogen-specific Igs has been applied in severalstudies [12, 72, 116, 126–128]. In fact, as observed byTrevisi et al. [12] the trend of total serum IgA did notreflect the trend of F4-specific IgA; thus, the analysis oftotal IgA rather than specific IgA could mask interestingresults regarding the specific response of the piglets tothe infection. The different response between total orpathogen-specific IgA could be due to the fact that totalIgA production can be stimulated by the bystander acti-vation of B cells caused, for instance, by the LPS. Thisbystander stimulation improved B cell mitosis and in-duced a polyclonal response, increasing the productionof a non-specific antibody in a T-cell dependent or inde-pendent manner [129].It should be noted that neither the ELISA kit nor F4

and F18 specific antigens are commercially available.However, the protocols for determining specific ETECF4 and F18 have been published [72, 126]. These proto-cols involve the collection of F4 and F18 fimbriae to beprepared for analysis of the specific F4/F18 fimbrial anti-gens in a blood sample.Differences in the immune response to ETEC F4 and

F18 inoculation can be observed. The synthesis ofF4-specific IgA is faster and more intense thanF18-specific IgA, which can be ascribed to the higher

Fig. 3 Increment of serum F4-specific immunoglobulin A (IgA) in piglets after enterotoxigenic Escherichia coli (ETEC) F4ac inoculation. Barsrepresent the fold change of F4-specific IgA in serum between the pre-challenge and the post-challenge period. * data were transformed fromlog2 values. Dpi: days post-inoculation


ability of ETEC F4 to adhere on the brush border com-pared to ETEC F18 [72]. Specifically, the serumF4-specific IgA increased from 4 to 7 dpi, and reachedlog2 6 titers [72] and its level stayed at this high leveluntil 14–18 dpi [12, 13]. Several studies have observedthat F4-specific IgA titers increased from 310% to 662%in the period from the pre-challenge to 4–5 dpi andreached an increase of 857% at 7 dpi (Fig. 3). SerumF18-specific IgA increased at 11 dpi and reached a max-imum level at 21 dpi when its amount was reported aslog2 4 [72].The serum level of specific IgA can be affected by pig-

let priming and by the individual amount of F4/18Rs onthe brush border [89, 126].Similar to the plasma or serum concentration of IgA,

blood IgM and IgG displayed differences in timing andquantification between F4 and F18 ETEC inoculation.F4-specific IgGs in the blood started to increase at 4 dpiand achieved a plateau at 7 dpi while the F18-specificIgGs increased only after 11 dpi and reached their max-imum level at 25 dpi. The F4-specific IgMs started to in-crease at 4 dpi and had their maximum level at 7 dpiwhile the F18-specific IgMs only slightly increased until7 dpi and then decreased from 15 dpi [72].In addition to blood serum Ig qualification, some studies

developed protocols for Ig quantification in saliva andfeces [84, 89, 130]. The application of non-invasivemarkers in an ETEC challenge study can be of notableinterest to promote the refinement approach in vivo stud-ies. Fecal immunoglobulin quantification has frequentlybeen used in humans to assess intestinal permeability, in-testinal epithelial barrier functionality and bacterial trans-location [131]. In pigs, fecal immunoglobulins have onlybeen scarcely investigated. In the study of Rossi et al. [89],the quantification of fecal IgA coupled with health param-eters allowed assessing the piglets’ response to ETECinoculation after vaccinations. The quantity of fecal IgA isinfluenced by age and by passive immunity received fromthe sow [130, 132]; thus, these factors need to be takeninto account in longitudinal studies which use fecal IgA asan immunological marker. In addition, fecal IgA can varyaccording to host-microbiota interaction [133]; therefore,commensal bacteria other than the inoculated ETEC canaffect the fecal IgA titer. To overcome this inaccuracy,specific fecal F4 and F18 IgA should be analyzed in ETECchallenge studies, as proposed for porcine epidemic diar-rhea virus infection [134].Saliva sampling is easy to carry out and is stress-free;

however, very little information has been reported re-garding Ig salivary kinetics after ETEC inoculation. Theexisting information is limited to IgA class and to stud-ies using the F4 challenge model. With respect toblood F4-specific IgA, a lower level of F4-specificIgA is reported in the saliva [135]. Its level increases

after the challenge up to 7 dpi [128]; however, a de-scription of their kinetics over time is lacking. Con-trary to the differences in blood F4-specific IgAbetween susceptible and resistant piglets, no geneticdifference in F4-specific IgA is observed in saliva[136]. Some authors have suggested that the lack ofdifference in saliva IgA between susceptible and re-sistant piglets could be due to a local mechanism ofthe immune response from salivary glands or tosampling issues [137].

Expression of the ETEC-specific receptor in the intestinalmucosaThe genotyping for the different markers associated withthe ETEC susceptibility reported in the previous para-graph increased the likelihood of identifying ETECF4- and F18-susceptible piglets. However, the phenotypicexpression of the receptors, especially the F4R, has alarge variability, and is believed to involve gene epistasis[58]. Therefore, to confirm piglet ETEC susceptibility, itis necessary to assess the expression of F4/F18 receptorson the intestinal brush border. Protocols to evaluate thepresence of ETEC receptors consist of a post-mortem invitro adhesion test which has been developed for bothETEC F4 and F18. This in vitro test consists of countingthe number of ETEC F4 or F18 adhering bacteria on thebrush border of the jejunum villi. Detailed protocols areexplained by Van den Broeck et al. [126] for ETEC F4adhesion, and by Verdonck et al. [74] and Yokoyama etal. [114] for the ETEC F18 adhesion. As an alternativemethod, an ex vivo approach has been proposed by Sugi-harto et al. [138] which consists of an intestinal organculture (PIOC) of ETEC and subsequent ETEC plateenumeration.Overall, the authors observed that 12 out of the 48

studies carried out a post-mortem confirmation of pig-lets’ susceptibility to the inoculated ETEC strain. The re-sults obtained were used by the authors to confirm theanimal’s susceptibility to ETEC (presence or absence ofreceptors) or to classify the animals based on their ETECsusceptibility (number of receptors per unit of villi sur-face [126]). In the latter case, the authors used the invitro adhesion test data as an individual scoring of pigletsusceptibility; the scoring was then used to classify theanimals (mildly or highly susceptible) and it was addedas a factor in the statistical model [116, 139]. However,no difference between homo and heterozygous suscep-tible genotypes to ETEC was obtained with regard to thelevel of intestinal adherence of ETEC measuredex vivo [138].

Conclusion and perspectivesThe literature review pointed out the differences in thepiglets’ response to F4 and F18 inoculation, especially in


terms of the intensity and the timing of the diarrheaand of the piglets’ immunological response and theirpathogen fecal shedding. Additional research is neces-sary to assess the piglets’ response to ETEC F18 in-oculation in order to define the timing and the valuesof the indicators for the development of the challengemodel. Table 4 summarizes the main features whichneed to be taken into account when designing anETEC challenge trial, including the setting of the modeland the criteria which allow a correct evaluation of thechallenge effectiveness. The wide individual response vari-ability observed among piglets to the ETEC challenge canbe partially controlled by proper choice (based on geneticmarkers) and assessment (with the analysis of ETECreceptors) of ETEC-susceptible animals. Inclusion of

pathogen-specific indicators such as specific F4 and F18Igs, ETEC F4/F18 fecal enumeration and the in vitro ETECadhesion test would be desirable to properly justifythe effect of the specific interventions when the chal-lenge model is applied. The above are important forthe optimization of the experimental design and, inthis way, take into consideration the 3R approachwhen using the piglet challenge model, especially con-cerning the issues Reduction and Refinement.

AbbreviationsACK1: Tyrosine kinase, non-receptor, 2; B3GNT5: UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 5; BPI: Bactericidal/permeability-increasing protein; CFU: Colony-forming unit; DM: Dry matter; Dpi: Dayspost-inoculum; E. coli: Escherichia coli; ETEC: Enterotoxigenic Escherichia coli;F4/18R: F4/18 receptors; FUT1: Alpha (1,2)-fucosyltransferase;Ig: Immunoglobulin; LAB: Lactic acid bacteria; MUC4/ MUC13/

Table 4 Main features for assessing an ETEC challenge trial, including the setting of the model and the criteria for evaluation of thechallenge effectiveness

Setting of the challenge model

Animal selection

Genetic markers which assist inETEC susceptibility

ETEC F4: non coding region [54]; MUC4 [48]; MUC13 [49, 50] ETEC F18: FUT1 [60]; BPI [62]

Sanitary status of the farm Previous ETEC infection on the farm; Vaccine prophylaxis

Suggested animal preconditioning Treatment with narrow-spectrum antibiotics

Fasting for 3 h + 62 mL of a 1.4% NaHCO3-solution

Control group Negative control group -- > recommended if a feed interventionis predicted to influence the PWD via immunological mechanisms

Timing of the inoculum 4–7 dpw -- >multifactorial stress of weaning followed by a dropin passive immunity

Inoculation method and dosage Oral administration: to comply with the Refinement approach

ETEC F4: 1×108 CFU/mL - 1.5 mL of 1010 CFU/mL ETEC F18: 5 mL of 108 CFU/mL - 10 mLof a 1011 CFU/mL

Evaluation of the challenge effectiveness

Diarrhea and related indicators Fecal score: daily from a day before ETEC inoculation until thepigs recovered

Diarrhea incidence: peak during the first week post-ETECinoculation

Days with diarrhea: daily from a day before ETEC inoculationuntil the pigs recovered

Fecal DM: daily from a day before ETEC inoculation until the pigsrecovered: 12.9–20.4% during dpi 1 to 3

Rectal temperature 39.0–39.5 °C pre-challenge to > 40.0 °C 6 h’ post-inoculation,then decreasing gradually

Bacterial fecal shedding Evaluation of ETEC F4 and F18 fecal shedding in the period frombefore inoculation and 3–4 dpi

ETEC F4: 2 dpi (5.97 × 108 F4 per gram of feces), then decreasingsuddenly

ETEC F18: 3–5 dpi (9.9 × 107 F18 pergram of feces), then decreasinggradually

Immunoglobulins Secretory IgA: mucosal and/or bile samples

F4-specific and F18-specific IgA from blood plasma at 5 dpi

Expression of ETEC specific receptorsin the intestinal mucosa

Post-mortem in vitro adhesion test

Intestinal organ culture (PIOC)

Abbreviations: ETEC Enterotoxigenic Escherichia coli, MUC4 Mucine 4, MUC13 Mucine 13, FUT1 Fucosyltransferase 1, BPI Bactericidal/permeability increasing protein,PWD Post-weaning diarrhea, dpw Days post-weaning, DM Dry matter, dpi Days post-inoculum, IgA Immunoglobulin A, PIOC Intestinal organ culture


MUC20: Mucin4, Mucin13, Mucin20; PIOC: Porcine intestinal organ culture;PWD: Post-weaning diarrhoea; RT: Rectal temperature; SIgA: Secretory IgA;TFRC: Transferrin receptor

AcknowledgementsThis study is based upon work from COST Action FA1401, supported byCOST (European Cooperation in Science and Technology).

FundingNot applicable.

Availability of data and materialsAll data generated or analysed during this study are included in this publishedarticle [and its supplementary information files].

Authors’ contributionsAll authors designed and wrote the study. All authors edited and approvedthe manuscript.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Agricultural and Food Sciences (DISTAL), Alma MaterStudiorum - University of Bologna, Bologna, Italy. 2Faculty of Science andTechnology, Department of Animal Science, Aarhus University, Tjele,Denmark.

Received: 17 December 2018 Accepted: 4 April 2019

References1. COMMISSION IMPLEMENTING DECISION of 26.6.2017 concerning, in the

framework of Article 35 of Directive 2001/82/EC of the European Parliamentand of the Council, the marketing authorisations for veterinary medicinalproducts containing “zinc oxide” to be administered orally to foodproducing species. 2017. Available from: https://ec.europa.eu/health/documents/community-register/2017/20170626136754/dec_136754_en.pdf.

2. Suiryanrayna MVAN, Ramana JV. A review of the effects of dietary organicacids fed to swine. J Anim Sci Biotechnol. 2015;6:45.

3. Zeng Z, Zhang S, Wang H, Piao X. Essential oil and aromatic plants asfeed additives in non-ruminant nutrition: a review. J Anim SciBiotechnol. 2015;6:7.

4. Roselli M, Pieper R, Rogel-Gaillard C, de Vries H, Bailey M, Smidt H, et al.Immunomodulating effects of probiotics for microbiota modulation, guthealth and disease resistance in pigs. Anim Feed Sci Technol. 2017;233:104–19.

5. Le Floc’h N, Wessels A, Corrent E, Wu G, Bosi P. The relevance of functionalamino acids to support the health of growing pigs. Anim Feed Sci Technol.2018;245:104–16.

6. Waititu SM, Yin F, Patterson R, Yitbarek A, Rodriguez-Lecompte JC, NyachotiCM. Dietary supplementation with a nucleotide-rich yeast extract modulatesgut immune response and microflora in weaned pigs in response to asanitary challenge. Animal. 2017;11(12):2156–64.

7. Che L, Hu L, Liu Y, Yan C, Peng X, Xu Q, et al. Dietary nucleotidessupplementation improves the intestinal development and immunefunction of neonates with intra-uterine growth restriction in a pig model.PLoS One. 2016;11(6):e0157314.

8. Zhang J, Li Z, Cao Z, Wang L, Li X, Li S, et al. Bacteriophages asantimicrobial agents against major pathogens in swine: a review. J Anim SciBiotechnol. 2015;6:39.

9. Triantafilou M, Triantafilou K. Lipopolysaccharide recognition: CD14, TLRsand the LPS-activation cluster. Trends Immunol. 2002;23(6):301–4.

10. Yao M, Gao W, Tao H, Yang J, Huang T. The regulation effects of danofloxacinon pig immune stress induced by LPS. Res Vet Sci. 2017;110:65–71.

11. Biswas SK, Lopez-Collazo E. Endotoxin tolerance: new mechanisms,molecules and clinical significance. Trends Immunol. 2009;30(10):475–87.

12. Trevisi P, Colombo M, Priori D, Fontanesi L, Galimberti G, Calò G, et al.Comparison of three patterns of feed supplementation with liveSaccharomyces cerevisiae yeast on postweaning diarrhea, health status, andblood metabolic profile of susceptible weaning pigs orally challenged withEscherichia coli F4ac. J Anim Sci. 2015;93(5):2225–33.

13. Trevisi P, Corrent E, Mazzoni M, Messori S, Priori D, Gherpelli Y, et al. Effectof added dietary threonine on growth performance, health, immunity andgastrointestinal function of weaning pigs with differing geneticsusceptibility to Escherichia coli infection and challenged with E. coli K88ac.J Anim Physiol Anim Nutr. 2015;99(3):511–20.

14. Fairbrother JM, Nadeau É, Bélanger L, Tremblay CL, Tremblay D, Brunelle M,et al. Immunogenicity and protective efficacy of a single-dose live non-pathogenic Escherichia coli oral vaccine against F4-positive enterotoxigenicEscherichia coli challenge in pigs. Vaccine. 2017;35(2):353–60.

15. Sugiharto S, Lauridsen C, Jensen BB. Gastrointestinal ecosystem andimmunological responses in E.coli challenged pigs after weaning fed liquiddiets containing whey permeate fermented with different lactic acidbacteria. Anim Feed Sci Tech. 2015;207:278–82.

16. Virdi V, Coddens A, De Buck S, Millet S, Goddeeris BM, Cox E, et al. Orally fedseeds producing designer IgAs protect weaned piglets against enterotoxigenicEscherichia coli infection. Proc Natl Acad Sci U S A. 2013;110(29):11809–14.

17. Sugiharto S, Hedemann MS, Lauridsen C. Plasma metabolomic profiles andimmune responses of piglets after weaning and challenge with E. coli. JAnim Sci Biotech. 2014;5(1):17.

18. Clark JM. The 3Rs in research: a contemporary approach to replacement,reduction and refinement. Br J Nutr. 2018;120(s1):S1–7.

19. Dubreuil JD, Isaacson RE, Schifferli DM. Animal enterotoxigenic Escherichiacoli. EcoSal Plus. 2016;7(1). https://doi.org/10.1128/ecosalplus.ESP-0006-2016.

20. Ravi M, Ngeleka M, Kim SH, Gyles C, Berthiaume F, Mourez M, et al.Contribution of AIDA-I to the pathogenicity of a porcine diarrheagenicEscherichia coli and to intestinal colonization through biofilm formation inpigs. Vet Microbiol. 2007;120(3–4):308–19.

21. Moredo FA, Piñeyro PE, Márquez GC, Sanz M, Colello R, Etcheverría A, et al.Enterotoxigenic Escherichia coli subclinical infection in pigs: bacteriologicaland genotypic characterization and antimicrobial resistance profiles.Foodborne Pathog Dis. 2015;12(8):704–11.

22. Zajacova ZS, Faldyna M, Kulich P, Kummer V, Maskova J, Alexa P. Experimentalinfection of gnotobiotic piglets with Escherichia coli strains positive forEAST1and AIDA. Vet Immunol Immunopathol. 2013;152(1–2):176–82.

23. Loos M, Geens M, Schauvliege S, Gasthuys F, Van Der Meulen J, Dubreuil D,et al. Role of heat-stable enterotoxins in the induction of early immuneresponses in piglets after infection with Enterotoxigenic Escherichia coli.PLoS One. 2012;7(7):e41041.

24. Luppi A. Swine enteric colibacillosis: diagnosis, therapy and antimicrobialresistance. Porcine Health Manag. 2017;3:16.

25. Jacobs AA, Venema J, Leeven R, van Pelt-Heerschap H, de Graaf FH.Inhibition of adhesive activity of K88 fibrillae by peptides derived from theK88 adhesin. J Bacteriol. 1987;169(2):735–41.

26. Jin LZ, Marquardt RR, Zhao X. A strain of enterococcus faecium (18C23)inhibits adhesion of enterotoxigenic Escherichia coli K88 to porcine smallintestine mucus. Appl Environ Microbiol. 2000;66(10):4200–4.

27. Van den Broeck W, Cox E, Oudega B, Goddeeris BM. The F4 fimbrial antigenof Escherichia coli and its receptors. Vet Microbiol. 2000;71(3–4):223–44.

28. Francis DH, Erickson AK, Grange PA. K88 adhesins of enterotoxigenicEscherichia coli and their porcine enterocyte receptors. Adv Exp Med Biol.1999;473:147–54.

29. Grange PA, Erickson AK, Levery SB, Francis DH. Identification of an intestinalneutral glycosphingolipid as a phenotype-specific receptor for the K88adfimbrial adhesin of Escherichia coli. Infect Immun. 1999;67(1):165–72.

30. Melkebeek V, Rasschaert K, Bellot P, Tilleman K, Favoreel H, Deforce D, et al.Targeting aminopeptidase N, a newly identified receptor for F4ac fimbriae,enhances the intestinal mucosal immune response. Mucosal Immunol. 2012;5(6):635–45.

31. Tusell SM, Schittone SA, Holmes KV. Mutational analysis of aminopeptidasen, a receptor for several group 1 coronaviruses, identifies key determinantsof viral host range. J Virol. 2007;81(3):1261–73.

32. Coddens A, Valis E, Benktander J, Ångström J, Breimer ME, Cox E, et al.Erythrocyte and porcine intestinal glycosphingolipids recognized by F4fimbriae of Enterotoxigenic Escherichia coli. PLoS One. 2011;6(9):e23309.


https://ec.europa.eu/health/documents/community-register/2017/20170626136754/dec_136754_en.pdfhttps://ec.europa.eu/health/documents/community-register/2017/20170626136754/dec_136754_en.pdfhttps://doi.org/10.1128/ecosalplus.ESP-0006-2016

33. Grange PA, Mouricout MA, Levery SB, Francis DH, Erickson AK. Evaluation ofreceptor binding specificity of Escherichia coli K88 (F4) fimbrial adhesinvariants using porcine serum transferrin and glycosphingolipids as modelreceptors. Infect Immun. 2002;70(5):2336–43.

34. Rippinger P, Bertschinger HU, Imberechts H, Nagy B, Sorg I, Stamm M, et al.Designations F18ab and F18ac for the related fimbrial types F107, 2134Pand 8813 of Escherichia coli isolated from porcine postweaning diarrhoeaand from oedema disease. Vet Microbiol. 1995;45(4):281–95.

35. Francis DH. Enterotoxigenic Escherichia coli infection in pigs and itsdiagnosis. J Swine Health Prod. 2002;10(4):171–5.

36. Wittig W, Klie H, Gallien P, Lehmann S, Timm M, Tschäpe H. Prevalence ofthe fimbrial antigens F18 and K88 and of enterotoxins and verotoxinsamong Escherichia coli isolated from weaned pigs. Zentralbl Bakteriol. 1995;283(1):95–104.

37. Nagy B, Whipp SC, Imberechts H, Bertschinger HU, Dean-Nystrom EA, CaseyTA, et al. Biological relationship between F18ab and F18ac fimbriae ofenterotoxigenic and verotoxigenic Escherichia coli from weaned pigs withedema disease or diarrhoea. Microb Pathog. 1997;22(1):1–11.

38. Nagy B, Fekete PZ. Enterotoxigenic Escherichia coli in veterinary medicine.Int J Med Microbiol. 2005;295(6–7):443–54.

39. Smeds A, Hemmann K, Jakava-Viljanen M, Pelkonen S, Imberechts H, PalvaA. Characterization of the adhesin of Escherichia coli F18 fimbriae. InfectImmun. 2001;69(12):7941–5.

40. Coddens A, Verdonck F, Tiels P, Rasschaert K, Goddeeris BM, Cox E. The age-dependent expression of the F18+ E. coli receptor on porcine gut epithelialcells is positively correlated with the presence of histo-blood groupantigens. Vet Microbiol. 2007;122(3–4):332–41.

41. Nagy B, Fekete PZ. Enterotoxigenic Escherichia coli (ETEC) in farm animals.Vet Res. 1999;30(2–3):259–84.

42. Peterson JW, Whipp SC. Comparison of the mechanisms of action ofcholera toxin and the heat-stable enterotoxins of Escherichia coli. InfectImmun. 1995;63(4):1452–61.

43. Kyriakis SC, Tsiloyiannis VK, Vlemmas J, Sarris K, Tsinas AC, Alexopoulos C, etal. The effect of probiotic LSP 122 on the control of post-weaning diarrhoeasyndrome of piglets. Res Vet Sci. 1999;67(3):223–8.

44. Trevisi P, Latorre R, Priori D, Luise D, Archetti I, Mazzoni M, et al. Effect of feedsupplementation with live yeast on the intestinal transcriptome profile ofweaning pigs orally challenged with Escherichia coli F4. Animal. 2017;11(1):33–44.

45. Spitzer F, Vahjen W, Pieper R, Martinez-Vallespin B, Zentek J. A standardisedchallenge model with an enterotoxigenic F4+ Escherichia coli strain inpiglets assessing clinical traits and faecal shedding of fae and Est-II toxingenes. Arch Anim Nutr. 2014;68(6):448–59.

46. Girard M, Thanner S, Pradervand N, Hu D, Ollagnier C, Bee G. Hydrolysablechestnut tannins for reduction of postweaning diarrhea: efficacy on anexperimental ETEC F4 model. PLoS One. 2018;13(5):e0197878.

47. Coddens A, Loos M, Vanrompay D, Remon JP, Cox E. Cranberry extractinhibits in vitro adhesion of F4 and F18+ Escherichia coli to pig intestinalepithelium and reduces in vivo excretion of pigs orally challenged withF18+ verotoxigenic E. coli. Vet Microbiol. 2017;202:64–71.

48. Jørgensen CB, Cirera S, Anderson SI, Archibald AL, Raudsepp T, ChowdharyB, et al. Linkage and comparative mapping of the locus controllingsusceptibility towards E. coli F4ab/ac diarrhoea in pigs. Cytogenet GenomeRes. 2003;102(1–4):157–62.

49. Ren J, Yan X, Ai H, Zhang Z, Huang X, Ouyang J, et al. Susceptibility towardsEnterotoxigenic Escherichia coli F4ac diarrhea is governed by the MUC13gene in pigs. PLoS One. 2012;7(9):e44573.

50. Zhang B, Ren J, Yan X, Huang X, Ji H, Peng Q, et al. Investigation of theporcine MUC13 gene: isolation, expression, polymorphisms and strongassociation with susceptibility to enterotoxigenic Escherichia coll F4ab/ac.Anim Genet. 2008;39(3):258–66.

51. Ji H, Ren J, Yan X, Huang X, Zhang B, Zhang Z, et al. The porcineMUC20 gene: molecular characterization and its association withsusceptibility to enterotoxigenic Escherichia coli F4ab/ac. Mol Biol Rep.2011;38(3):1593–601.

52. Wang Y, Ren J, Lan L, Yan X, Huang X, Peng Q, et al. Characterization ofpolymorphisms of transferrin receptor and their association with susceptibilityto ETEC F4ab/ac in pigs. J Anim Breed Genet. 2007;124(4):225–

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