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Shiga/Verocytotoxins and Shiga/ verotoxigenicEscherichia coli in animals

Jacques Mainil

To cite this version:Jacques Mainil. Shiga/Verocytotoxins and Shiga/ verotoxigenic Escherichia coli in animals. VeterinaryResearch, BioMed Central, 1999, 30 (2-3), pp.235-257. <hal-00902568>

Review article

Shiga/Verocytotoxins and Shiga/verotoxigenic Escherichia coli in animals

Jacques Mainil

Chairc de bactoi-iologie ct pathologie des maladies bact6riennes, faculte de medecine v6t6rinaire,univcrsité de Liege. Sart Tilman, Bat. B43a, B-4000 Liege, Belgium

(Received 19 October 1998; accepted 21 January 1999)

Abstract - Vero/Shiga toxins (VT/Stx) have an A-B structure: the A subunit can-ies the enzymaticactivity and the B subunit binds the toxin to the membrane receptor (Gb3 or Gb4). The VT/Stxinhibit protein synthesis in the target eucaryotic cells, mainly the endothelial cells of blood vessels.The VT/Stx arc subdivided into two families. VT I /Stx] is a homogeneous family of toxins identicalto the Stx of Shigella dysenleriae. VT2/Stx2 is a more heterogeneous family of toxins more dis-tantly related to this Stx toxin. The VT2/Stx2 variants can be distinguished by the polymerase chain nreaction (PCR) and/or the reaction with monoclonal antibodies. The VT/Stx-producing Escherichiacoli are also subdivided into two main groups on the basis of the presence or absence of additional prop-ei-lies: the enterohaemorrhagic E. coli (EHEC) induce the formation of attaching/effacing lesionsand carry a 60 MD plasmid encoding a specific haemolysin (the enterohaemolysin); the vero/shiga-toxigenic E. coli (VTEC/STEC) do not show these properties. The EHEC are isolated from humansand ruminants, especially young calves. They are associated with haemorrhagic enterocolitis andits sequelae in humans, the haemolytic-uracmic syndrome (HUS). The VT/Stx play a role in theoccurrence of blood in the faeces and in the HUS by their action on the endothelial cells of blood ves-sels in the intestinal submucosa and in the renal glomeruli, after resorption through the intestinalwalls. The VTEC/STEC are isolated from piglets, calves and humans. In recently weaned piglets, theycause the oedema disease, an enteiotoxaemia characterized by subcutaneous, inesenteiic and cerebraloedemas, with nervous disorders as main clinical signs. The oedema disease is the consequence of theaction of the VT/Stx on the endothelial cells of blood vessels in various organs. In calves and humans,the role in disease of VTEC/STEC is controversial, but they could be associated with some cases ofdiarrhoea and HUS. The case of the 0157:H7 EHEC which are present in healthy cattle of various ages,but are highly virulent for humans is of special interest. The potential zoonotic aspect of VT/Stx-pro-ducing E. coli infections in animals is detailed chapter by chapter. Prophylaxis of these infections byvaccination is the subject of the discussion on the future of the research studies on these pathogenicbacteria. O Inra/Elsevicr, Paris.

E. coli / shigatoxins / verotoxins

Tel.: (32) 4 366 40 50; fax: (32) 4 366 41 22; e-inail: jg.muinilC!ulg.ac.bc

Résumé - Cytotoxines Shiga/Vero et Escherichia coli Shiga/Vérotoxinogènes chez les animaux.Les toxines Véro/Shiga (VT/Stx) sont composées de deux sous-unités : la sous-unité A, siège del’activité enzymatique, et la sous-unité B, qui se fixe sur le récepteur membranaire (Gb3 ou Gb4). Lestoxines VT/Stx agissent par inhibition de la synthèse protéique dans les cellules eucaryotes cibles, essen-tiellement les cellules endothéliales des vaisseaux sanguins. Les toxines VT/Stx produites par E.sche-richia coli se répartissent en deux familles : VTI/Stx 1, une famille homogène de toxines identiquesà la toxine Stx de Shigella dysenteriae, et VT2/Stx2, une famille plus hétérogène de toxines pluséloignées de cette toxine Stx. Les différents variants de la famille VT2/Stx2 peuvent être distinguéspar amplification génique en chaîne (PCR) et/ou réaction avec des anticorps monoclonaux. Lessouches d’E. coli productrices de toxines VT/Stx sont subdivisées en deux grands groupes sur basede l’existence ou non de propriétés annexes : les souches entérohémorragiques (ECEH) induisent laformation de lésions d’attache men t/efface ment et possèdent un plasmide de 60 MDa qui code pourune hémolysine spécifique (I’entérohémolysine) ; les souches Véro/Shigatoxinogènes (ECVT/ECST)ne possèdent pas ces propriétés. Les souches ECEH se retrouvent chez les humains et les ruminants,principalement les jeunes veaux. Elles sont associées à des entérocolites hémorragiques et à sesséquelles éventuelles chez l’homme, comme le syndrome héinolytique-urémique (SHU). Les toxinesVT/Stx joueraient un rôle dans l’aspect hémorragique de la diarrhée ainsi que dans le développementdu SHU, par action sur les cellules endothéliales des vaisseaux sanguins de la paroi intestinale et desglomérules rénaux, après résorption intestinale. Les souches ECVT/ECST se retrouvent chez lesporcelets, les veaux et les humains. Chez des porcelets récemment sevrés, elles sont responsables dela maladie de l’oedème, une entérotoxémic caractérisée par l’apparition d’oedèmes sous-cutanés,mésentérique et cérébral, avec comme signes cliniques principaux des troubles nerveux. La maladiede l’eedème est la conséquence de l’action des toxines VT/Stx sur les cellules endothéliales des vais-seaux sanguins de divers organes. Chez le veau et l’homme, le rôle des souches ECVT/ECST est parcontre controversé, mais elles pourraient être associées à certains cas de diarrhées et de SHU. Un casparticulier est celui des souches ECEH 0157:H7 présentes chez des bovins en bonne santé de tous âges,mais très virulentes pour l’homme. L’aspect zoonotique potentiel des diverses infections à E. coli pro-ductrices de toxines VT/Stx est d’ailleurs détaillé chapitre par chapitre. Quant à la prophylaxie de cesinfections par vaccination, elle fait l’objet de la discussion sur les perspectives des recherches concer-nant ces bactéries pathogènes. &copy; Inra/Elsevier, Paris.

E. coli / toxines Shiga / toxines Véro

1. INTRODUCTION

1.1. Historical background

In 1977, Konowalchuk et al. [69]reported a toxin active on cultured Verocells, [hence the name verocytotoxin (VT) ]produced by a dozen human and porcinediarrhoea- and edema disease-associatedEscherichia coli. The cytopathic effect ofVT is quite different from the non-cyto-pathic effect of the classical heat-labileenterotoxin of enterotoxigenic E. coli(ETEC). The same year, O’ Brien et al. [92 ]reported that several E. coli, including thehuman H30 strain also studied byKonowalchuk et al. [69], are cytotoxic forcultured HeLa cells. This cytotoxicity isneutralized by an immune serum specificfor the Shiga toxin (Stx) from Shigelladysenteriae type 1, hence the name Shiga-like toxin (SLT) [93].

These groups of cytotoxins are hetero-geneous because the toxicity of severalhuman (H.1.8, E32511, 933, etc.) andporcine (E57, P104) strains is not, or onlypartially, neutralized by immune seradirected to the toxin of the human strainH30 and to the Stx of Shigella dy.sentericaetype l. This is in contrast to the toxicity ofother strains [61, 69, 117, 128]. The lattercytotoxins are identical to the Stx of Shigelladvsenteriae and were renamed VT1 or

SLT 1, whereas the former toxins receivedthe name VT2 or SLT2.

1.2. Nomenclature

The discovery of these cytotoxins by dif-ferent research teams has resulted in a par-allel nomenclature system. The terms ’vero-toxins (VTs)’ or ’Shiga-like toxins (SLTs)’on the one hand and ’verotoxigenic E. coli(VTEC)’ or ’Shiga-like toxin-producing E.coli (SLTEC)’ on the other have been usedfor many years by Canadian/British workersand American workers, respectively,although they are synonyms. More recently,the proposal to use the names ‘Shiga toxins(Stx)’ for all these cytotoxins was made [191,but did not receive unanimous approval [62].

The main arguments in favour of the

newly proposed nomenclature are thatVT/SLT are identical or related to the Stx of

Shigella dy.seoteriae and that the descrip-tion of the Stx precedes the description ofVT/SLT. The main argument in favour ofmaintaining the original nomenclature ofVT is based upon the fact that not all VTsare identical to the Stx of Shigella dysente-riae (VT1 are, but the various VT2 are not)and therefore distinct names should be used.VT/Stx must thus be considered as syn-onyms and throughout the text, the newlyproposed nomenclature will be used.

Another confusing area concerns thenomenclature of the various Stx-producingE. coli. Basically three groups are described:- those associated with haemorrhagic col-

itis (HC) and its sequelae, the haemolytic-uraemic syndrome (HUS) in humanbeings (which, in addition to the pro-duction of Stx, are also able to inducethe ’attaching-effacing’ (AE) lesions).They belong to a few main serotypes(0157:H7, 0157:H-, 026:H) 1, 026:H-,0111:H-, Ol 13:H21, 0103:H2, etc.),and possess a ca. 60 MDa plasmid encod-ing for a specific enterohaemolysin [89,95, 139];

- those showing basically the same prop-erties, but isolated from animals, mainlycalves, and causing enteritis and bloodydiarrhoea [ I 8, 75, 143];

- those not showing these properties, iso-lated from human beings, sometimes inassociation with HUS [89, 95, 139J, frompigs as a cause of edema disease [4, 40,139, 143J, and from other animal specieswith or without clinical signs [16, 75, 96,139p.Strains from the first group are referred to

as ’enterohaeinorrhagic E. coli’ or EHEC,although bloody diarrhoea is not systemat-ically observed. The name EHEC was atone time restricted to the strains belongingto the O 157 serogroup. Similar strains fromanimals are referred to as EHEC or EHEC-like isolates, even if they are not especiallyassociated with haemorrhagic colitis. If theauthor agrees with these two definitions andnames, he does not, however, agree withthe most often proposed name for the strainsfrom the third group, i.e. EHEC also or

’atypical EHEC’ [891. Indeed it is his feel-ing that this name does not suit strains fromthis third group, especially porcine strainswhich are not basically associated withintestinal disorders [41. The author will usethe name STEC (Shigatoxigenic E. coli) forthem, which best describes their main prop-erty, i.e. the production of Stx, until othervirulence factors or virulence-associated

properties are described.

1.3. Structure of the manuscript

After a presentation of the most recentmolecular data on Shiga toxins, the cliniccal, bacteriological and epidemiological dataon Stx-producing E. coli will be extensivelyreviewed in ruminants and in pigs. After ashort note on Stx-producing E. coli in otheranimal species, the past and future history ofShiga toxins will be discussed. A review onhuman Stx-producing E. coli would havebeen justified because of the public healthproblems and because the Stx-producing E.coli from animals are almost always studiedin comparison with their human counter-parts. However, a lot of current literaturefocuses on them and the role of Stx-pro-ducing E. coli in human infections and dis-eases (diarrhoea, haemorrhagic colitis andsystemic complications, such as the

haemolytic-uraemic syndrome) has thusbeen reviewed recently by several authors[89, 95, 97, 1391. Moreover, the most recentepidemiological and clinical data on 0157strains are covered in a recently publishedbook [59]. There is thus no need to repeatwhat has already been presented and dis-cussed several times.

2. THE SHIGATOXINSOF ESCHERICHIA COLI

Extensive and numerous reviews on the

Shiga toxin (Stx) families have been pub-lished since their description. This chapter isbased on the most recent ones [83, 89, 95,114]. Moreover, the recently published bookmentioned above presents the most recentcellular and molecular data on Stx [59!. Afew specific and/or even more recent refer-ences have been added in the text.

2.1. The Shiga toxin family

As already mentioned, the Stx producedby E. coli consist of two families: Stx I, ahomogeneous group of toxins virtually iden-tical to the Stx of Shige lla dysenteriae type

1 and Stx2, a heterogeneous group of toxinsmore distantly related to the Stx. The pro-totype Stxl is produced by the E. coli strainsH 19, H30 and 933 [69, 93, 117, 128 Vari-ants have been described which only differby one amino acid with no consequence ontoxicity or on antigenicity.

The prototype Stx2 is produced by E coli-type strains 933 and E32511 [61, 114, 128 1.The first Stx2 variants were actuallydescribed by Konowalchuk et al. [69], butwere rediscovered a few years later [ 117 1,and characterized even later. The Stx2e toxinis produced by E. coli-type strains E57,S 1 191 and 412 associated with the edemadisease in recently weaned piglets [50, 81,135] and occasionally by strains of otherorigins [44, 98, 105, 129, 136J. Other namessuch as Stx2v (after variant) or Stx2vp (aftervariant pig) exist. The other Stx2 variantdescribed by Konowalchuk et al. [69] is pro-duced by the human E. coli strain H.1.8. It ismore closely related to Stx2e than to Stx2and is named Stx2ev, after edema diseasevariant (or Stx2va, or Stx2vh after varianthuman) [47]. The Stx2ev has not beendescribed for other strains.

Several other Stx2 variants produced byhuman and animal E. coli have been and arestill being described [2, 99]. Amongst them,the Stx2c variant produced by the E. coliE3251 I strain [ 1 14] appears to be the mostepidemiologically and clinically importantin humans. These variants were recognizedon the basis of partial neutralization by anti-Stx2 immune serum and/or the absence of a

positive PCR reaction. E. coli can produceStx I or Stx2 only, or both (strain 933 f.i.), orStx2 and a Stx2 variant, mainly the c variant(strain E32511 f.i.), or two Stx2 variants.

2.2. Structure and biological activities

Stx have an A-B subunit structure andconsist of one A subunit of approximately33 kDa and five B subunits of approximately7.5 kDa each. The A subunit is the biologi-cally active portion of the toxin and the B

subunits bind the toxin to the glycolipidmembrane receptor, globotriaosylceramideor Gb3 (Galal-4Gal) for Stxl and Stx2, butglobotetraosylceramide or Gb4 (GalNac! 1-3Galal-4Gal) for Stx2e and Stx2ev. Othervariants have intermediate affinity for thesereceptors. Receptor specificity is based on afew amino acids in the B subunit.

Internalization of the toxins is achieved

by receptor-mediated endocytosis followedby transport to the Golgi apparatus and thento the endoplasmic reticulum. The A sub-unit is translocated into the cytoplasm andcleaved to yield a 28 kDa N-terminal peptide(A 1 ) with the enzymatic activity and a 4 kDaC-terminal peptide (A2). The A I peptideacts as an N-glycosidase by removing anadenine residue from the 28S rRNA of the60S subunit of eucaryotic ribosomes, there-fore preventing elongation-factor-1-depen-dent binding of aminoacyl-tRNA to ribo-somes. The end result is the inhibition of

protein synthesis leading to cell death.

The range of biological activities of Stxinclude cytotoxicity, enterotoxicity and neu-rotoxicity.

An observation already reported byKonowalchuk and Speirs [68 and con-firmed a few years later is that all Stx are

highly toxic in vitro for Vero cells but notnecessarily for other cells [14, 64]. Stxl andStx2 are also toxic for HeLa cells. Stx2e is10 000 times less toxic and other Stx2 vari-ants are 10-100 times less toxic for HeLacells than for Vero cells. The situation is the

opposite for MDBK cells. These observa-tions are related to the amount of specificreceptors present on the cells: mainly Gb3(receptor for Stx 1 and Stx2) on HeLa cells,mainly Gb4 (receptor for Stx2e) on MDBKcells, similar levels of Gb3 and Gb4 on Verocells. In vivo, the target cells are mainly theendothelial cells in the kidneys, brain and/orstomachal and intestinal submucosa accord-

ing to the infected species. However, in the-ory, any cell which possesses the appropri-ate receptor(s) at its surface may be a targetcell.

Stx I and Stx2 are also considered to be

enterotoxic, i.e. to cause fluid accumulationin ligated intestinal segments in rabbits, suchas the heat-stable and heat-labile entero-toxins of ETEC. Although this effect hasnot been observed by all authors, it may berelated to the various Stx produced. In con-trast the Stx2e toxin is poorly (100 timesless) enterotoxic. The mechanism of this

enterotoxicity is still not completely under-stood and the following explanations areconsidered:

- selective destruction of the absorptivevillus tip enterocytes which present highlevels of Gb3 receptor in their membranein contrast to the secretary crypt entero-

cytes;- inhibition in the absorption of NaCi, with-

out any effect in ion secretion, especiallyC I-;

- damage to the underlying vessels by adirect effect on endothelial cells with lossof fluids and cells into the gut lumen;

- production of another toxin which isenterotoxic, such as the EAST toxin, orenteroagregative heat stable toxin, pro-duced by enteroaggregative E. coli [ l 11 j.

Neurotoxicity of Stx has been observedrepeatedly in mice and rabbits parentallyinoculated with extracts of Sh. d i7,veiiteriaeand of Stx-producing E. cmli. It is illustratedby an ascendant paralysis and by lethality.This neurotoxicity is most probably a con-sequence of the damage caused by the tox-ins to the vascular endothelial cells of thecentral nervous system. Neurotoxicity hasalso been reproduced in piglets during stud-ies on edema disease and is thus demon-strable for all types of Stx.

2.3. Genetics and production

Stx are encoded by an operon (st-vAB)with two open-reading frames for the A andB subunits. The yt.YA and .s/.fB genes formone transcriptional unit. The Stx-producing

E. coli can possess one stx operon (stxl or.stx2) or two .stx operons [stxl and .stx2 (strain933 [57]), or stx2 and one stx2 variant (strainE32511 1 [ 1 1 4 ] ), or two stx2 variants (strainB2F1 [56J!. ].

The stxl operons are identical or veryclosely related to the .stx operon of Shige!ladv.senteriae type 1. As already said the Stx2are more distantly related to the Stx toxinof Shigella dysenteriae type I, so that thestxIAB and stx2AB prototype operons only yshare 57 % nucleotide sequence identity intheir st.vA and 60 % in their .st.xB genes.Although Stx I and Stx2 differ in their genesencoding the B subunits, they have the samereceptor specificity, Gb3.

Phylogenetic trees based on the publishednucleotide sequence of the A and B sub-

unit-encoding genes of the stx2 operon vari-ants have been constructed showing the exis-tence of clusters [3, 99, 1 15 The .stx2operon variants are closely related to eachother within each cluster (>95 °Io overall

identity) and more closely related to the .stx2operon (74-96 % overall identity) than tothe stxl operon (55-60 % overall identity).The st.x2e and stx2ev operons are the most

distantly related to the .stx2 operon with 94and 70 % identity, respectively, in the genesencoding the A subunit and only 78/79 %in the genes encoding the B subunit. Thisexplains their different receptor specifici-ties (Gb4).

Originally O’Brien et al. 1931 describedtwo groups of Stx-producing E. coli: thoseproducing high levels of toxins detectablein the culture supernatants and those pro-ducing low levels of toxins detectable onlyin bacterial extracts. Otherbacterial speciescould also produce high or low levels oftoxins f 1 1, 93, 113, I 20]. The stx genes havebeen detected by DNA hybridization and/orPCR only in the E. coli and in other bacterialspecies (Citrobacterfreundii and Enter-obacter spp.) producing high levels of tox-ins. They have, however, never beendetected in the E. coli nor in the other bac-terial species producing low levels of toxins.

The stxl, ,stx2 and the stx2c operons arelocated on lambdoid phages in E. colibelonging to various serotypes, especiallyO157, whereas the .s?A operon in Sfii,q«lladysenterine, the stx2e and possibly the other.ctx2 variant operons in E. coli are not. Thetransfer of .stxl and stx2 operons to recipientstrains can be performed in vitro by phageconversion and is suspected in vivo [2, 1 l6,122, I 25 ],

The production of the Stx I and Stx of E.coli and Sh. dysenteriae, but not of Stx2 andof the Stx2 variants of E. coli, is regulated atthe transcriptional level and is repressed bya high concentration of iron and by a lowtemperature. The production of the Stx2eby porcine strains is repressed in the wild-type bacteria by another still unknownmechanism.

After their synthesis the Stx are exportedto the periplasm by a type II signalsequence-mediated secretion system. Theirmain localization seems to be the periplas-mic space, especially for Stx l , whereas Stx2and the Stx2 variants appear to be more eas-

ily excreted into the culture medium.

2.4. Detection and diagnosis

The Stx can be detected in the super-natants of bacterial cultures and in biologi-cal samples such as faeces. In bacterial cul-ture, even higher titres can be obtained aftertreatment to lyse the cells [93! or bypolymyxin B !37] to release the cell-boundtoxins present in the periplasmic space.

Three types of assays are basically used:cell toxicity assays, immunological assaysand genetic assays.

The cell toxicity assays are of course wcll lknown 169, 93!. They are still consideredas the most sensitive, especially when Verocells arc used, and can be applied on cul-ture supernatants, on cell extracts and also

directly on faccal samples after the adapta-tion of the protocols. The use of Vero cellsis recommended to detect all variants. Oth-

erwise the use of other cells is to be con-sidered (HeLa, MDBK f.i.). Primary cellcultures, such as endothelial cells, are alsosensitive to the Stx but are not used for rou-tine detection assays. Cells are observed for

any cytopathic effect for 3 days and the iden-tity to one of the so far described Stx is per-formed by neutralization with specificimmune serums. There is no cross-reactionbetween Stx I and Stx2 and only partiallybetween Stx2 and most variants.

Several immunoassays have been devel-oped to detect Stx in culture supernatants, inbacterial extracts, or directly in faecal sam-ples [89, 95]: sandwich and other ELISAassays with immune serums, monoclonalantibodies or a Gb3 receptor as a capturesystem and the reverse passive latex agglu-tination assay. Only the use of some spe-cific monoclonal antibodies allows us to

fully distinguish between the different Stx2variants. Interestingly enough, Stx-produc-ing Pseudomonas aeruginosa strains cancause false positive reactions in a commer-cial enzyme immunoassay for Stx [) 1]. I.Other Stx-producing bacteria may thus rep-resent a cause of false positive reactions.

The genetic assays are subdivided intothe colony hybridization assays with polynu-cleotidic or oligonucleotidic probes and thepolymerase chain reaction (PCR) assays.The colony hybridization assay with polynu-cleotidic probes was the first one to be used[90 The distinction between .stxl and stx2and variants is easy to perform with any ofthe probe systems. Most of the probesderived from one .stx2-related operon cross-

hybridize with all str2 operon variants. Itis, however, possible to use oligonucleotideprobes and especially PCR assays to achievea differcntial detection of the Stx2 variants.It is also possible that other bacterial speciesharbouring sfx-related genes represent acause of false positive reactions in hybridiza-tion assays, as they can in enzymeimmunoassays.PCR systems have been developed to

detect the presence or sfx operons, either ontheir own or along with the detection of

other genes in multiplex PCR reactions [3,25, 43, 99, 115]. Of the many primersdesigned some detect all stx (stxl and thevarious stx2) operons, others are specificfor the stxl operons, for all the stx2 vari-

ants, or for only one of them. The PCR tech-niques are very sensitive and specific forthe characterization of bacterial isolates, butare not as good for the characterization offaecal samples, because of the presence ofnon-specific inhibiting factors in the faeces.For faeces they are regarded as less sensitivethan cell toxicity assays.

3. STX-PRODUCING ESCHERICHIACOLI AND RUMINANTS

Stx-producing E. coli present two kindsof interactions with cattle: i) intestinal dis-eases in newborn to 4-month-old calves; ii)carriage by healthy animals, young calvesand adults. In addition to cattle, Stx-pro-ducing E. coli are also detected in the faecesof other domestic and wild ruminants eitherin association with disease or in healthy ani-mals. The pathology of Stx-producing E.coli in cattle has been the object of reviewsfor several years [18, 75, 100, 136, 139,143]. In contrast, the carrier state has beenintensively studied only for the last 3 or 4years and recent reviews are referred to [2,31,53,89].

3.1. Clinical conditions in young calves

The first reports of production of Stx byE. coli isolated from diarrhoeic calves were

published in 1980 [63, 140]. The cytotoxinof the strain isolated by Kashiwazaki et al.[63] was antigenically similar to the cyto-toxin of strain H30 [64] and was thus amember of the Stx 1 family. The associationbetween diarrhoea in calves and the pres-ence of Stx toxin-producing E. coli was con-firmed in 1985 [51, 85, 120]. During thefollowing years, a number of reports werepublished which in addition brought infor-

mation about the general and specific prop-erties and characteristics of these bovine

Stx-producing E. coli.

Stx-producing E. coli are associated withdiarrhoea in 1-8-week-old calves with a

peak between 4 and 5 weeks of age,although newborn and older calves can alsobe affected [27]. The diarrhoea observed ismucoid, sometimes haemorrhagic and dif-fers from the aqueous diarrhoea caused byETEC. Diarrhoea does not cause the death ofthe calf very often (the case fatality rate doesnot exceed a few per cent), but it is recurrenteven after treatments, leading to dehydra-tion, weakening and a depressed growthrate. Necropsy lesions of enteritis are mainlylocalized in the large intestine with the pos-sibility of extension to the small intestinein severe cases. Localized and diffuse haem-

orrhages can be observed. In severe cases,other diarrhoeagenic infectious agents aredetected: cryptosporidia, rotavirus, coron-avirus, coccidia and ETEC in very youngcalves.

Systemic complications, such as HUS,have never been observed in calves. Themost probable reason for this is the absenceof specific receptors on kidney glomerularendothelial cells (see section 2.2).

3.2. Serotypes and pathotypes of Stx-producing E. coli in young calves

Most of the Stx-producing E. coli asso-ciated with diarrhoea in young calves belongto the following serotypes: 05:H-, 08:H8,020:HI9, 026:Hll, 0103:H2, Ol 11:H-,0111:H8, OI11:H11, 0118:H16 and0145:H+. Some of these serotypes are alsorecovered from humans (see section 3.5).Other serotypes are detected much less fre-quently, but many strains are either non-typable with the immune sera used or roughvariants. The 0157 strains have only excep-tionally been associated with clinical dis-eases [ 15, 94].

The bovine Stx-producing E. coli can besubdivided into EHEC and STEC (see def-initions in section 1.2). The former mainlybelong to the 05, 026, 0103, O111, 0118 8and 0145 serogroups and are associatedwith clinical diarrhoea and enteritis. Mostof the latter belong to the 08 and 020serogroups. The situation of STEC is equiv-ocal as some authors regard them as non-pathogenic because they are frequently iso-lated from healthy as well as from diarrhoeiccalves. In some studies STEC are, however,clearly isolated in cases of diarrhoea. BovineSTEC may thus represent a heterogeneouspopulation of bacteria [104, l07], only someof which are pathogens because they pos-sess additional still uncharacterized viru-lence-associated properties.

3.3. Virulence propertiesand pathogenesis in young calves

In addition to the production of Stxbovine, EHEC and STEC indeed possessother properties which may be related totheir pathogenicity. The data on bovineEHEC and STEC are reviewed in compari-son with the properties of the human strains.

3.3.1. The Stx toxins

The majority of bovine EHEC associatedwith diarrhoea in young calves produce (orhybridize with a specific gene probe) Stxl Ionly and only a few Stx2, or both toxins.However, bovine STEC from young calvesare positive for Stxl and/or Stx2 [79, 137 1.The Stx2 produced are either the prototypetoxin or a variant, especially Stx2c, but alongstill untyped variants [77, 123, 137]. Inhuman EHEC the Stxl, Stx2 and Stx2c canbe phage-encoded [115]. Localization of thestx operons on phages has also been reportedor suspected in bovine EHEC and STEC [2,109].

Since the first publication associatingStx-producing E. coli with HC and HUS inhumans, there have been numerous studies

and debates over the exact role and mecha-nism of action of the toxins. Studies usingdifferent animal models have sometimesfurther confused the debate because the sig-nificance of Stx can vary according to themodel used. After comparison of the wild-type EHEC with their isogenic mutants inthe stx genes, a general agreement isreached: the Stx play no role in the devel-opment of the enteritis and in the occurrenceof diarrhoea but are important in the haem-orrhagic aspect of HC and in the develop-ment of HUS. A comparison of the wild-type and isogenic mutant strains in a calfinfectious model would most probably con-firm that in calves the Stx are not involvedin the occurrence of diarrhoea but rather inthe haemorrhagic aspect of the enteritiswhen present. Other properties must thusbe expressed by the bovine EHEC andSTEC to explain the diarrhoea.

3.3.2. The attachingleffacing (AE)phenotype

Bovine EHEC are capable of causing AElesions and this phenotype is considered torepresent the primary cause of the diarrhoeaobserved [51, 106, 107]. Indeed E. coli pro-ducing AE lesions but not Stx

(enteropathogenic E. coli or EPEC) are bythemselves a cause of diarrhoea in humansand in different animal species. Productionof AE lesions is a multistep event charac-terized by: i) initial adherence to themicrovilli of the enterocytes; ii) transduc-tion of a signal into the enterocytes; iii)cytoskeleton rearrangement with effacementof the microvilli of the enterocytes; and iv)intimate attachment to the enterocyte cyto-plasmic membrane !84, 89].

The initial adhesin of bovine EHEC, if

any, still awaits identification, although can-didates [144] and positive adherence prop-erties to appropriate cells of bovine origin inculture [138] have been described. The

genetic determinants for the last three stepsare, however, better known. These genesare grouped together on the chromosome

forming one pathogenicity island calledLEE, for the locus of enterocyte effacement.The LEE of bovine EHEC (and EPEC)show similarities and differences in com-

parison to the LEE of human EHEC and/orEPEC. The genes differing between humanand bovine EHEC could serve as epidemi-ological markers in public health studies(see section 3.5) [26, 48, 136].STEC strains which can be isolated from

clinical cases are AE phenotype-negative.If STEC are diarrhoeagenic E. coli, theymust possess other properties which causethe occurrence of diarrhoea. These proper-ties have not yet been characterized butsome are suspected (see section 3.3.4).

3.3.3. The enterohaemolysin andthe ’EHEC virulence plasmid’

Human 0157 and many non-0157EHEC harbour a large plasmid called the’EHEC virulence plasmid’ [60]. It carriesgenes encoding one enterohaemolysin, i.e. ahaemolysin active only on washed red bloodcells (ehx genes), for a type II secretion sys-tem (etp genes), for a catalase-peroxydase(katP gene), and for a secreted serine pro-tease (espP and pssA genes). Its involve-ment in the initial attachment of EHEC is,however, still controversial. The role ofthese properties in the survival of the bac-teria and/or in the development of the dis-ease is purely speculative.

Different enterohaemolysins (Ehly) havebeen described in E. coli, but this one is rel-atively specific for EHEC, from which thename EHEC Ehly comes [73]. The EHECEhly belongs to the RTX (repeat in toxins)family of toxins and ca. 60 % identity wereobserved between the ehx and hly (encodingthe a-haemolysin of E. coli) genes. Thepresence of the ’EHEC virulence plasmid’can be detected using gene probes and PCRsfor the various systems described [60]. Theehx and etp genes are present in all 0157EHEC, whereas the katP and espP genesare detected in two-thirds of them. In non-0157 EHEC, the ehx genes are present in

almost all strains tested, the etp in half ofthem, and the others in only a third of them.

The majority of bovine EHEC and STECproduce the enterohaemolysin phenotypeon washed sheep blood cell agar plates ortest positive with the pCVD419 probe whichcorresponds to the ehx genes. The EHECenterohaemolysin phenotype or genotype iscorrelated with the production of Stxl aloneor with Stx2 (at least 75 % of the strainstested), but less often with the productionof Stx2 alone (50 % of the strains tested)[103, 118, 136, 137]. The production ofenterohaemolysin by bovine EHEC andSTEC is also mediated by a plasmid [136,137]. Whether this plasmid carries the othergenetic determinants present on the ’EHECvirulence plasmid’ of human EHEC is yetunknown.

3.3.4. Others

Human 0157 and non-0157 EHEC andSTEC can produce the heat-stable toxin ofenteroaggregative E. coli (EAST 1) (112]whose role in pathogenesis is speculative,but which may be responsible in part for theoccurrence of non-bloody diarrhoea. BovineEHEC and STEC have not been tested forthe EAST1, to our knowledge.

Bovine EHEC and STEC are generallyresistant to tellurite (Pohl and Mainil, unpub-lished data) as are human O 157 EHEC andShig>ella spp. [ 145]. The relevance to thepathogenesis of this resistance is question-able. This resistance might play some role inthe pathology of those strains and/or in theirecology and survival in the cattle rumen andgut and in the environment. It is most usefulfor their selection by incorporating telluritecompounds in growth media 195 1.

The strains belonging to the 05:H-serotype possess very rare properties for E.coli, i.e. they produce no gas during carbo-hydrate fermentation and are capable of fer-menting urea [22, 102]. The genetic deter-minism of these properties has, to ourknowledge, never been studied.

3.4. Carrier state in healthy cattle

Most cases of HC and HUS in humansare caused by ingestion of foods and drinkscontaminated with faeces from cattle, espe-cially ground beef, undercooked hamburg-ers, salami or other foods sometimes of cat-tle or small ruminant origin (raw milk orhome-made cheese from raw milk), but alsonon-pasteurized apple cider and juice,uncooked vegetables and water from wellor municipal systems. Less frequent modesof transmission of the infection are cattle-to-

person and person-to-person direct contacts[89, 95, 97, 139]. These observations haveprompted large scale surveys, over the pres-ence of EHEC and STEC, especially of the0157 serogroup, in beef and dairy herds ofhealthy adult cattle, in healthy young calves,and at slaughter in young bulls, culled cowsand veal calves. The surveys were per-formed in European countries (Belgium,France, Germany, Holland, Italy, Spain, UK,etc.), in North America (Canada and USA),in South America (Chile, etc.), and in Asia(Thailand, Sri Lanka, etc.) [2, 27, 53, 89,105].

Stx-producing E. coli were detected inall surveys in most if not in all herds, butthe proportion of positive animals in herdsvaried greatly from study to study and some-times reached 100 %. Many of the positivestrains detected belong to serotypes com-mon among human EHEC and STEC. Geo-

graphical, management and seasonal fluc-tuations are possible explanations for thevariable prevalence of EHEC and STEC ofthe different serotypes, but most probablythe population sampling and the techniquesof detection account for most of the varia-tions. 0157:H7 and 0157:H- EHEC have amuch lower, but also highly fluctuable,prevalence on farms and at slaughter, ascompared to other serotypes: from less than0.5 % up to 10 % of positive animals.

The Stx-producing E. coli from healthyanimals may represent non-pathogenicstrains, or strains pathogenic for humans butnot for cattle, or strains pathogenic for calves

(although the conditions for the develop-ment of the disease were not present at thetime of sampling). One way of answeringthis question is to develop challenge exper-iments in calves. Experimental infectionshave been conducted with 0157 humanEHEC. They confirmed that 0157 EHECare not pathogenic for calves aged 1 weekand more [17, 28], but also demonstratedpathogenicity for newborn calves [30, 31]. ].In older animals 0157:H7 EHEC behavemuch as commensal E. coli more than

pathogenic E. coli with a short-term colo-nization (<2 months) of the rumen and/orthe intestine and shedding [53).

Another way of answering the abovequestions is to compare the general and spe-cific properties of the Stx-producing E. colifrom healthy cattle to those of Stx-produc-ing E. coli associated with clinical disease inhumans and in calves.

3.5. Epidemiology, sources of infectionand public health hazard

The epidemiology of EHEC and STECinfection in young calves has not been fullystudied but it seems reasonable to speculatethat they are both carried by healthy indi-viduals, calves and adults, and that the clin-ical disease is triggered by managementproblems such as overcrowding, transport,presence of other pathogens, etc. Diseasedcalves and healthy cattle can be infectedwith more than one strain based on serotype,pathotype, and the pulse field gel elec-trophoresis (PFGE) profile ([2, 12, 104,107], China and Mainil, unpublishedresults). ).

The main problem is, however, the pub-lic health concern about the transmission ofbovine EHEC and STEC to humans. Toanswer that question a comparison of humanand bovine strains must be carried out usingthe same epidemiological tools.

Identical serotypes and pathotypes havebeen detected for human and bovine EHEC,

f.i. 026:H11, 0103:H2, 0111:H-, and0157:H7 [89, 95, 97, 136, 137, 139]. Someof these strains could be pathogenic forhumans and calves (026, O 103, 01 I I),whereas others would be pathogenic onlyfor humans (0157). More precise molecularstudies of the bacteria and of their virulence-associated factors and properties (stx genesand phages, EHEC virulence plasmids, LEE,tellure resistance, etc.) must be carried out tofully compare these bacterial strains andgroup them according to their pathogenicspecificity and public health hazard poten-tial. A recent study has detected differencesin the LEE of the majority of EHEC (andEPEC) associated with diarrhoea in calvesand of those isolated from healthy animals[27].

3.6. Diagnosis

The diagnosis and characterization ofEHEC and STEC in cattle follow the same

general approach as with human strains [89,95!: cell cultures, ELISAs and/or PCRs areused for the Stx and/or the other propertieseither on bacterial isolates or directly onfaeces. Specific ELISAs and uni- or multi-plex PCRs on bovine faeces have beenrecently designed [1, 2, 27, 43]. Positive E.coli can be subsequently serotyped (see sec-tion 3.2). For detection of O 157 EHEC car-riers, special methods with enrichment andmagnetic bead separation are being usedmuch as the methods used in food microbi-

ology [89, 95].

3.7. Infections in other ruminants

Small ruminants have been the subjectof fewer surveys than cattle until recentlywhen 0157 EHEC were detected in sheepand goat in faeces or at slaughter [ 13, 24,54, 70], thus showing that small ruminantsmay also represent a source of contamina-tion for humans. Sheep positive for Stx-pro-ducing E. coli of other serotypes have alsobeen detected with a wide variation from

flock to flock [9, 10, 38, 58, 71]. In goatflocks, the percentage of positive animalsalso varied greatly, from 7 to 95 % [2, 9,10]. Positive strains have various Stx profilesand many hybridize with the pCVD419 9probe for the ehx genes. Transmission ofO 157 and other serotype EHEC to humans

by raw goat milk or home-made raw-milkcheese have been demonstrated [13, 20].0157 EHEC have also been isolated fromwild and farmed deer in the UK and USAand were associated with a human outbreakin one case [23, 65, 108]. Stx-producing E.coli of various serogroups have also beendetected in diarrhoeic and non-diarrhoeicbuffalo calves in Sri Lanka [85].

4. STX-PRODUCING ESCHERICHIACOLI AND PIGS

Besides cattle and humans, Stx-producingE. coli are also associated with a well-doc-umented disease in piglets, the ’edema dis-ease’ (ED), whose first published clinicaldescription dates from 1938 in Ireland [119].Although many clinical studies were car-ried out already in the 1950s, it was notbefore the 1980s that the most importantbacteriological and molecular data wereobtained.

The pathogenesis of ED and the viru-lence factors of the associated Stx-produc-ing E. coli are the subjects of several reviews[5, 40, 49, 55, 139, 143]. Most pertinentdata are summarized below along with afew additional references.

4.1. Clinical conditions

ED occurs most generally as sudden out-breaks in recently weaned piglets (1-2weeks after weaning). Many affected pigletsare found dead while others present nervousclinical signs (ataxia, convulsions) alongwith subcutaneous edema, especially on thehead (eyelids, frontal area, groin). Diarrhoeais only rarely observed in true edema dis-

ease. Morbidity can reach 30-40 % in somefarms with a case fatality rate higher than90 %. Sporadic cases have also beendescribed in younger suckling piglets andin older sows and boars.

At necropsy, the bodies are in good con-dition with a full stomach. Edema of thesubcutaneous tissue, mesentery, stomachsubmucosa (cardiac region) and spiral colonsubmucosa are the most remarkable fea-tures. Enteritis is present only in the ani-mals with clinical diarrhoea.

The typical histological lesions areextravasation and haemorrhages in variousorgans, mainly the central nervous system,the stomach, the large intestine and the sub-cutaneous tissue.

4.2. Serotypes and pathotypes

No more than four serotypes of E. coliare responsible for the great majority of theclinical outbreaks of ED throughout theworld: 045:K+ (type strain E65), 0138:K81 1(type strain E57), 0139:K82 (type strainE4), and 0141:K-, formerly 0141:K- (typestrains E68II, E145) [126]. The 0141:K85serotype is also associated with neonataland post-weaning diarrhoea.

The porcine Stx-producing E. coli belongto the STEC pathotype. EHEC strains havebeen observed only once and no clinicaldata were available [80]. In addition to Stx,the strains associated with diarrhoea (post-weaning diarrhoea or PWD) produce clas-sical enterotoxin(s) and/or the fimbrialadhesin K88(F4) of enterotoxigenic E. coli(STEC/ETEC strains) [52, 143].

4.3. Virulence propertiesand pathogenesis

The main virulence factors of ED-asso-ciated E. coli are the Stx2e toxin and theF18 fimbrial adhesins acting as coloniza-tion factors. Some accessory virulence fac-tors have also been described.

4.3.1. The Stx2e toxin

Speculation about the production of atoxic factor in the pathogenesis of ED camefrom early experimental reproduction of theclinical signs by intravenous injection ofbacteria-free fluid from the intestinal con-tents of affected animals [130, 131]. Thistoxic factor received different names accord-

ing to its properties in different experimen-tal models: edema disease toxin, neurotoxin,angiotoxin, etc. [4].

Only 20 years later Konowalchuk et al.[69] reported the production of a verocyto-toxin by several E. coli including strain E57.The E57 verocytotoxin activity was not neu-tralized by an immune serum to the Stxl ofstrain H30 [14,64,69]. Dobrescu [36] iden-tified this verocytotoxin with the edema dis-ease toxin. The pig verocytotoxin is actu-ally a variant of the Stx2 family (STx2e,previously Stx2vp), with differences in itsbiological activity (no activity on HeLa cells,no enterotoxicity) and partial neutralizationby immune serums to Stx2 (see sections 2.2and 2.3).

The Stx2e described so far are homoge-neous and with a very few exceptions arepig specific [44, 98, 105, 129, 136]. Theonly related variant is produced by thehuman strain H.I.8 and has been namedStx2ev (for edema disease variant) [47]. Stxother than Stx2e (Stx 1, Stx2) have onlyexceptionally been detected in porcine E.coli not associated with ED [45, 46, 72, 76].

Stx2e plays a central role in the patho-genesis of ED and in the occurrence of clin-ical signs. After colonization of the gut,STEC produce Stx2e which crosses theintestinal epithelium and reaches the bloodstream. From there Stx2e reaches its target,i.e. the Gb4 receptor present on the endothe-lial cells of small arteries and arterioles invarious tissues and organs causing themicroscopic lesions which are the basis oftypical macroscopic lesions and clinicalsigns. Clinical disease can be reproducedby intraveneous administration of purified

Stx2e [74]. STEC are also associated withPWD, but Stx2e plays no role in the occur-rence of the diarrhoea which is caused byclassical enterotoxin(s) (STEC/ETECstrains) [52].

4.3.2. The colonization factors

ED-associated STEC produce no AElesions but rather fimbrial adhesins adhere tothe microvilli of the enterocytes of the smallintestine. A specific fimbrial adhesin, calledF 107 at first, was not described before the1990s because its production in vitro wasvery difficult to obtain [5]. Optimal expres-sion is obtained by growing the bacteria onblood agar under microaerobic conditions

[ 141 j. F107 antigenic variants produced bystrains associated with PWD were described:0141 fimbriae [66], 2134P [21, 88], F8813 3[III], but the existence of only two vari-ants was later suggested [ 14 1 F107ab, cor-responding to the variant produced by theED-associated E. coli, and F107ac, corre-

sponding to the variant produced by thePWD-associated E. coli. These fimbrial anti-

gens have now been renamed F] 8ab andFl8ac [110]. In vitro and in vivo expres-sion of Fl8ab by ED-associated STEC isgenerally poorer as compared to the expres-sion of Fl8ac by PWD-associated STEC[88a]. The F]8 fimbrial adhesin-encodinggenes are also present in non-toxigenic E.coli [80, 86].

The genes encoding the F18 fimbrialadhesins are located on plasmids, sometimesalong with genes encoding the a haemolysinand the classical enterotoxins of ETEC ([29,141 ], Mainil and Remy, unpublished data).The F18 fimbrial adhesin acts as a colo-

nization factor of the piglets small intestine(midjejunum and ileum) by mediating adher-ence of the STEC to the enterocytemicrovilli without any alteration of the

eukaryotic cells. The STEC subsequentlyproliferate and produce high amounts ofStx2e. Clinical observations of the 1950sof genetic and age resistance to ED are infact scientifically based upon the resistance

of the piglets to STEC colonization. Thegenetic resistance is due to the lack of spe-cific receptors to the Fl 8 fimbrial adhesin onthe enterocyte cytoplasmic membrane. Theproduction of specific receptors is controlledby one genetic locus and the susceptiblephenotype is dominant [6]. The suscepti-bility of the small intestine to colonizationby F18+ E. coli actually increases with age[88]. This observation is most probablyrelated to the absence of receptors on the

enterocyte membrane of newborn and veryyoung piglets. The receptors for the Fl8aband Fl8ac variants are most likely to be thesame, but differ from the receptors for the F4fimbrial adhesin of ETEC [88a].

4.3.3. The a-haemolysin

ED-associated STEC produce an a-haemolysin [73, 127]. Although there is noevidence that the a-haemolysin contributesto the pathogenesis of STEC in ED its pro-duction is very convenient for diagnosticpurposes even if many haemolytic E. coliisolated from piglets are not STEC. How-ever, non-haemolytic porcine STEC haveonly rarely been described [46, 72, 101, 125,134].The genes encoding the a-haemolysin

are located on a plasmid in porcine STEC.Very often, this plasmid also carries genesencoding the F18 fimbrial adhesin and/orclassical enterotoxin(s)([29,141], Mainil andRemy, unpublished data).

4.3.4. Others

The production of Stx2e is not system-atically related to any other property suchas the production of a colicin or to any spe-cific biotype [46, 101 ].

As already mentioned porcine STEC canalso produce classical heat-stable (STa, STb)and heat-labile (LT) enterotoxins and theF4 fimbrial adhesin of ETEC. The F4 fim-brial adhesin can also act as a colonizationfactor of the small intestine in recentlyweaned piglets and the enterotoxins are

responsible for clinical diarrhoea observedin some outbreaks of ED [52, 142].

4.4. Epidemiology and sourcesof infection

ED occurs as unpredictable sudden out-breaks in groups of recently weaned pigletsand is the consequence of the proliferation ofhaemolytic STEC with the production ofhigh amounts of Stx2e in the intestinallumen. Haemolytic E. coli can, however, beisolated from healthy piglets in almost allpig farms with and without clinical mani-festations of ED. What are the differencesbetween the various haemolytic E. co/<&dquo;? And

what causes their proliferation at weaningtime?

The main conclusions of a large fieldstudy carried out by Deprez et al. [33, 34] ]are:

- most haemolytic E. coli in farms withED but only a few of those in farms with-out ED are STEC;

- the haemolytic STEC are much morecompetitive in vivo to suppress the com-mensal intestinal flora than are the other

haemolytic E. coli;- the proliferation of STEC in the intestines

of recently weaned piglets is mainly theconsequence of the sudden removal ofthe maternal lactogenic protection;

- the other changes at weaning time (diet,regrouping, etc.) can play an additionalrole.

These observations are potential expla-nations for the fact that ED is an endemictransmissible disease in some farms only,i.e. in those in which the STEC circulate.

4.5. Diagnosis

Diagnosis of ED is mostly clinical, basedon the age of the piglets, on the circum-stances of occurrence of the disease, on theclinical signs and on the lesions observed

at necropsy. Bacteriological confirmationis, however, often requested. A pure cultureof haemolytic E. coli is usually obtainedfrom the small intestinal content. However,haemolytic E. coli are also isolated fromhealthy and PWD piglets in farms with orwithout ED outbreaks. Pohl et al. [101]detected 21 STEC out of 23 haemolytic E.coli isolated from piglets with ED, 15 STECout of 25 haemolytic E. coli recovered fromhealthy piglets in farms with ED and onlysix STEC out of 35 haemolytic E. coli whenthey were recovered from healthy piglets infarms without ED.

Confirmation that the haemolytic E. colirecovered are STEC is thus necessary andcan be obtained by cell culture assays (Stx2eis toxic for Vero cells and not for HeLa

cells), by immunological and/or by geneticassays. Stx2e cross-reacts to some extent

with other Stx2 variants in immunologicalassays (see section 2.4). Only monoclonalantibodies can distinguish Stx2e from Stx2and the other Stx2 variants. Similarly, a geneprobe derived from the stx2e gene cross-hybridizes with other stx2 gene variants [78],but specific PCR for the different stx2 genevariants have been developed [3, 96, 99!. ].

4.6. Public health hazard

Similarities between edema and centralnervous disorders in piglets with ED and ina few humans with HUS on the one hand,and the isolation of Stx2e-producing STECfrom human beings [44, 98, 129] on theother, have raised the question of humancontamination with porcine STEC strains.However, porcine STEC serogroups havenot yet been observed in humans with HCand HUS. In addition, if other STEC

serogroups have been isolated from healthypiglets and from pork products, only a veryfew belong to the O 157 serogroup and noneare true EHEC [24, 139]. In conclusion,piglets, pork and pork products are not con-sidered as a source of contamination withSTEC and EHEC for humans.

5. STX-PRODUCING ESCHERICHIACOLI AND OTHER ANIMALSPECIES

5.1. Mammals

Stx-producing E. coli (EHEC and STEC)have also been isolated from pets such asdogs and cats, although these reports arerare and usually incompletely documented.A quite recent publication even presents evi-dence of dogs as vectors for human con-tamination with 0157:H7 EHEC [132]. Datawere reviewed by Broes [16] and Peeters[96] and are reviewed in this issue [7].

Horses may also act as vectors of0157:H7 EHEC [132). Although an agree-ment on the involvement of E. coli in intesti-nal disorders in horses exists, their generaland specific characteristics are still largelyunknown [96].

5.2. Birds

In addition to mammals Stx-producingE. coli have also been isolated from birds

(poultry [39, 41, 82J, seagulls [133] andferal pigeons [32]). Their association withdisease is uncertain but they may act as vec-tors for infection of domestic ruminants andhumans. The most recent data on pathogenicE. coli for birds are also reviewed in thisissue [35].

6. CONCLUSION: PAST ANDFUTURE OF THE SHIGA TOXINS

The history of Shiga toxins encompassesone century considering that the descriptionof the causal agent of bacterial dysentery,Shigella dvsenteriae, was published in I 898by Shiga [ 12 1 (cited in [91 !). But the mostintense period of research on Shiga toxins ofE. coli began only 20 years ago with theworks of Konowalchuk et al. and O’Brien etal. [69, 93]. Many subsequent research stud-ies demonstrate that history (and research) is

a perpetual (re)discovery of old observa-tions.

In 1971, Smith and Linggood [124]referred to the production by the human E.coli H 1 of a ’heat-labile enterotoxin’ whichwas, however, not neutralized by an immuneserum specific for the LT of ETEC. More-over, if the capacity of producing this toxinwas transferable in a conjugation experi-ment, as was the capacity of LT productionwhich is plasmid-encoded, no plasmid wasdemonstrable in the recipient strain. Expla-nations came later: H19 produces Stxl (notLT) whose encoding genes are located ona phage [69, 116, 125].

Speculations that the HUS syndrome inhumans was caused by an enteropathogenicE. coli after acquisition of a bacteriophagewas raised in the late 1960s by Kibel andBarnard [67] (cited in [94]). Interestinglyenough, retrospective studies have reportedthe existence of Stx-positive E. coli (EHECand STEC) in collections of bovine strains ofthe 1960s [8, 77, 79, 103].

Another conclusion from Shiga toxin his-tory is that an exception today can becomea general rule tomorrow.

In their initial work, Konowalchuk et al.[69] reported the production of differentverocytotoxins. Indeed two E. coli produceda verocytotoxin whose toxicity was not neu-tralized by an immune serum to the cyto-toxins of the other strains: strain E57 from a

piglet with ED and strain H.1.8 from aninfant with diarrhoea. Moreover, their rangeof cytotoxicity differed from the other cyto-toxins [68]. It was later demonstrated thatstrain E57 produces the Stx2e toxin andstrain H.1.8, the Stx2ev toxin.

Let us now take a prospective look intothe future [91]. Much has been learned dur-ing the last 20 years, but there are still areaswhich escape our knowledge. Of the manyquestions to be addressed in the futureresearch works on Stx, STEC and EHEC,the following two are very important (fromthe author’s point of view).

- What is the ecology of EHEC, especiallyof the 0157 serogroup, in cattle?

- What is the vaccination potential againstSTEC and EHEC in humans and in ani-mals?

These questions will not be answeredeasily, especially the former one which willneed the use of time-consuming animalmodels to study colonization and survival[30, 53]. To answer the latter question inhumans, one needs animal models to repro-duce HUS [87]. Stx produce intestinal, cere-bral and kidney lesions in rabbit and miceafter parenteral injections, but not the typi-cal HUS syndrome. Besides non-human pri-mate models, another potential animal modelfor HUS is the greyhound in which a clini-cal condition similar to HUS (the cutaneousand renal glomerular vasculopathy, orCRGV) has been linked with 0157:H7 [42,87].

The situation is quite different in pigletsin which ED is reproducible by intravenousadministration of purified Stx2e and whichcan be protected in experimental and fieldtrials by vaccination with a purified toxoidor a mutant Stx2e [49].

The situation in cattle is two-fold. First,if EHEC are a cause of diarrhoea in youngcalves, the role of Stxl is still speculativeand no clinical systemic complications suchas HUS have been described. Vaccination

against EHEC thus targets the AE pheno-type more than the Stx [49]. There is alsomuch excitement and speculation about pre-venting the carriage of the 0157:H7 EHECby vaccination. It is, however, too early tosay that this will represent a powerful way ofpreventing carriage of 0157:H7 EHEC incattle [30,49].

ACKNOWLEDGEMENTS

The author wishes to thank first of all Pro-fcssor Albert Kacckenhecek. former head of the

department, without whom he would never haveworked on Escherichia cnli, and Dr Harley W.Moon (Iowa State University, Ames, Iowa,

USA), without whom he would never haveworked on Shigatoxigenic E.scherichia coli. Healso thanks his collaborators, Dr Bernard Chinaand Dr Fr6d6ric Goffaux, for animated and help-ful discussions and a long-term friend, Dr PierrePohl (Centre d’etude et de recherches vétéri-naires et agrochimiques, Brussels, Belgium),who recently retired, for critical reading of themanuscript. Finally he is endebted to Miss Vin-ciane Pirson and Mr Etienne Jacquemin, fromhis laboratory, for the collection of many refer-ences.

The research works on bovine ShigatoxigenicEscherichia coli in the author’s laboratory havebeen supported financially by the Institut pour]’encouragement de la recherche dans l’indus-trie et I’agriculture (research contract #5631 -1994-1996) and by the Minist!re des classesmoyennes ct de I’agriculture (research contract#5740 - i996-1998).

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