Enterotoxigenic Escherichia Coli Infection in Pigs and Its Diagnosis

Non refereed Diagnostic notes Enterotoxigenic Escherichia coli infection in pigs and its diagnosis

David H. Francis, PhD

nterotoxigenic Escherichia coli Table 1: Characteristics of enterotoxigenic Escherichia coli (ETEC) strains associated with infections in pigs of various ages1 (ETEC) is a major cause of illness and death in neonatal and recently E EdTEC characteristics Age group affecte weaned pigs. However, pigs older than ap- proximately 8 weeks appear to be resistant Seerogroup F)imbria Tsoxins(s Hemolysin 2 Ndeonatal Weane to infection. Strains of ETEC that cause diarrhea in pigs possess two types of viru- O98 Ka9 S–sT Yoe N lence factors, adhesins and enterotoxins, OP9 Ka99, 987 S–sT Yoe N both of which are essential for disease to OP20 9a87 S–sT Yoe N occur. O9101 Ka9 S–sT Yoe N Fimbrial adhesins OP141 9a87 S–sT Yoe N Porcine ETEC isolates produce any of five O88 Kb8 LT, ST ± S+Ta Yse sYe different adhesins, all of which are fimbriae O8149 Kb8 LT, ST ± S+Ta Yse sYe (also known as pili): K88 (F4), K99 (F5), 987P (F6), F41 (F7) and F18.1,2 Fimbrial O8157 Kb8 LT, ST ± S+Ta Yse sYe adhesins K88 and F18 occur in several an- Oc138 Fb18ab, F18a STa, ST ± S+tx2e Ns oYe tigenic forms. The K88 fimbrial variants Ob139 Fb18a STa, ST ± S+tx2e Ns oYe include K88ab, K88ac, and K88ad. By far the most prevalent, if not the only form of Oc141 Fb18a STa, ST ± S+tx2e Ns oYe K88 found in the United States, is K88ac.3 Oc157 Fb18a STa, ST ± S+tx2e Ns oYe Variants of F18 include F18ab and F18ac. Strains expressing F18ab may be more 1 Sources: Wilson RA, Francis DH, 1986;1 Imberechts H et al;4 and Diagnostic commonly associated with edema disease Laboratory Records, South Dakota State University Animal Disease Research and than strains expressing F18ac. Adhesins of Diagnostic Laboratory, Brookings, SD 57006 the F41 type are most often found in asso- 2 Presence (+) or absence (–) of hemolysins ciation with K99, and their significance in naturally occurring disease is uncertain. Enterotoxins Patterns of virulence The fimbrial adhesins target specific recep- Toxins produced by ETEC strains that tors on porcine intestinal brush border epi- determinants cause diarrhea in pigs include heat labile thelial cells (enterocytes), enabling the bac- Virulence determinants are not randomly teria to colonize the cell surface and there enterotoxin (LT), heat stable enterotoxin distributed among virulent strains, but excrete toxins whose action includes pro- type A (STa); heat stable enterotoxin type typically occur in patterns associated with duction of diarrhea in the host animal. Sus- B (STb); Shiga toxin type 2e (Stx2e), and specific serogroups and fimbriae. Cluster- ceptibility of pigs to ETEC is in large part enteroaggregative E coli heat-stable entero- ing of virulence determinants around 1,4,6 a function of expression and exposure of toxin 1 (EAST1). Enterotoxin Stx2e, serogroups, and the ages of pigs commonly these receptors on the intestinal luminal also known as edema disease factor, is the infected with ETEC of each virulence type, surface. Clinically apparent infection of cause of lesions associated with edema dis- are shown in Table 1. Almost all strains of pigs by ETEC is restricted both by animal ease in pigs. When absorbed into the ETEC isolated from weaned pigs with di- age and animal lineage.1,4,5 Lineage-re- blood, this toxin destroys endothelial cells arrhea contain the gene for STb. Near uni- stricted susceptibility of pigs to ETEC and, in small vessels, resulting in blood clots, versal presence of this gene in postweaning at least in some cases, age-restricted suscep- hemorrhage, ischemic necrosis, and edema diarrhea isolates strongly suggests that STb 7,8 tibility, is associated with availability of in vital organs, including the brain. The plays an important role in pathogenesis receptors for the attachment of fimbrial significance of EAST1 in porcine diarrheal that is not duplicated by other enterotox- 5 adhesins. disease is not known. ins. However, very few ETEC strains exhibit only STb genes. This suggests that Department of Veterinary Science, South Dakota State University, Brookings, SD 57007-1396. other enterotoxins are also important in This article is available online at http://www.aasv.org/shap.html. postweaning diarrheal disease. In addition to STb genes, postweaning isolates also Francis DH. Enterotoxigenic Escherichia coli infection in pigs and its diagnosis. J Swine Health Prod. 2002;10(4):171-175. typically possess genes for STa, LT, or both. Journal of Swine Health and Production — Volume 10, Number 4 171 Receptors for F18 fimbriae are not ex- Figure 1: The brush border adherence assay for determination of the + + pressed in neonatal animals and may not susceptibility-resistance phenotype of pigs to K88 and F18 enterotoxigenic + Escherichia coli. Bacteria with K88 or F18 fimbriae do not adhere to brush appear in sufficient numbers to allow F18 border vesicles prepared from enterocytes of pigs of the resistant phenotype ETEC infection until about the time when (left), but do adhere to brush border vesicles of pigs of the susceptible most pigs are weaned.14 Lack of suscepti- phenotype (right). (×1200) bility of neonatal pigs to F18+ ETEC strains probably is in consequence to a lack of F18 receptors. Patterns of lineage-associated susceptibility to various ETEC strains In addition to age-associated resistance of pigs to K88+ and F18+ ETEC strains, lineage-associated resistance to infection by these strains also exists. In each case, susceptibility is an autosomal dominant trait, and resistance is in consequence of failure of the animal to express the required receptors on enterocytes. When purebred pigs of four breeds (Chester White, Duroc, Hampshire, and Yorkshire) were tested for expression of K88 receptors, 46 of 96 animals (48%) expressed the receptors neces- Patterns of age-associated ceptibility of pigs to 987P+ ETEC.10 Re- sary for susceptibility to K88ac, the major susceptibility to various ceptors for K88 and F18 also appear to be K88 variant found in the United States.15 abundant in adult animals, again suggest- There was substantial variation in the ETEC strains ing that cessation of receptor expression is prevalence of susceptible animals among The vast majority of pigs clinically infected not the cause of age-associated resistance of the breeds, but the significance of this with ETEC strains expressing K99 or 987P older pigs to ETEC expressing these variation could not be determined due to are neonates, although pigs may be suscep- fimbriae.11 It is unlikely that age-associated the small sample size. The prevalence of tible to these strains for a week or more + + + pigs of the F18 susceptibility phenotype 1 resistance to 987P , K88 or F18 ETEC is after birth. Receptors for K99 fimbriae are in consequence to immunity developed has not been determined, but casual obser- displayed at diminished concentrations as from natural exposure to the organisms, as vation would suggest that it is considerably pigs mature beyond the neonatal stage, exposure to 987P+ ETEC and K88+ ETEC higher than that of K88 ETEC suggesting that susceptibility to ETEC ex- would likely occur at the same age, yet the susceptibility. pressing these fimbrial adhesins is a func- 9 age at which resistance is developed is dis- tion of receptor expression. Receptors for tinctly different for each strain. There is 987P appear to be expressed in abundance some evidence in mice for development of Figure 3: Intestinal impression in adult animals, suggesting that a lack of resistance to STa enterotoxin with age.12 smear from a pig infected with receptors is not the reason for loss of sus- Perhaps pigs also develop age resistance. enterotoxigenic Escherichia coli However, there seems to be little correla- stained by indirect immunofluorescence with anti- Figure 2: Hematoxylin-eosin tion between the type of enterotoxin pro- stained histologic section of small fimbria antibodies and fluorescein- intestine from a pig infected with duced by an ETEC strain and the age at isothiocyanate-conjugated enterotoxigenic Escherichia coli. which pigs become resistant. In addition, anti-immunoglobulin. Numerous Bacteria form a confluent layer on adult humans are susceptible to traveler’s fluorescing bacteria are present. the epithelial surface. (×400) diarrhea caused by ETEC. This indicates (×1200) that at least some adult animals are not resistant to all types of E coli enterotoxins. It is possible that changes in the intestinal brush border glycocalyx associated with animal aging render fimbrial receptors less available for bacterial attachment. As receptors for the different fimbrial adhesins are different in size and are probably different from each other in their presentation on the enterocyte surface, age-associated or diet-associated changes in the glycocalyx might mask receptors of different fimbriae at different animal ages.11,13

172 Journal of Swine Health and Production —July and August, 2002 K88 and F18 receptors on ceptibility-resistance phenotypes of pigs base pairs 307 and 857, which resulted in porcine enterocytes (Figure 1). A genetic marker of K88+ amino acid substitutions at positions 103 The receptor for K88ab and K88ac in por- ETEC resistance-susceptibility has not (threonine substituted for alanine) and 286 cine enterocytes has been identified as a been identified, although the resistance- (glutamine substituted for argenine). Ani- mucin-type sialoglycoprotein (IMTGP). susceptibility determinant appears to be on mals resistant to F18 ETEC are homozy- 16 This mucus-like molecule is anchored to chromosome 13. gous for threonine at amino acid 103 of 11 the FUT1 enzyme. Levels of FUT enzyme the brush border surface of enterocytes. The receptor for F18ab and F18ac has not are significantly lower in F18 ETEC-resis- Only pigs that express IMTGP are suscep- been identified, but a genetic marker for + + tible to K88ab and K88ac ETEC, and loss of receptor expression was shown tant animals, and Chinese hamster ovary + susceptibility of young pigs to K88 ETEC closely linked to the marker for porcine tissue culture cells transfected with the is highly correlated with expression of stress syndrome and porcine blood group FUT1 gene coding threonine at amino acid 5 18 IMTGP. The presence of IMTGP in por- inhibitor (S). The marker was mapped to 103 showed reduced enzyme activity. A cine enterocytes is also correlated with at- an alpha (1,2) fucosyltransferase (FUT) test for F18 receptor genotyping has been + + tachment of K88ab and K88ac bacteria gene on chromosome 6; namely, that for developed and worldwide license for the to brush border vesicles prepared from pig FUT1.17 Differences between susceptible test is held by the Pig Improvement Com- enterocytes collected by biopsy or at eutha- and resistant FUT1 gene open reading pany (Franklin, Kentucky). nasia. Thus, the brush border adherence frames (ORFs) were polymorphisms at assay is a useful test for K88+ ETEC sus-

Figure 4: Multiplex polymerase chain reaction (PCR) gel exhibiting positive control and Escherichia coli strains with typical and atypical fimbriae and toxin gene combinations. Lane 1: Base pair ladder used to estimate size of PCR products; Lane 2: Controls exhibiting PCR products of all genes identified on the left; Lane 3: F41+, K99+, STa+ strain; Lane 4: K88+, LT+, STb+ strain; Lane 5: Stx2e+ strain; Lane 6: F18+, STa+, STb+, Stx2e+ strain with a non-specific band between STx2e and F18; Lane 7: K88+, LT+, STa+, STb+ strain; Lanes 8 and 9- Strains exhibiting none of the virulence genes tested for; Lane 10: Blank.

Stx2e B41

K88 987P

F18 LT K99

ST a

ST b

Journal of Swine Health and Production — Volume 10, Number 4 173 smears prepared from cultures of the bacte- through a bacterial population. As many of Table 2: Genotypes of entero- 20, 21 toxigenic Escherichia coli strains ria (Figure 3). A potential pitfall in the virulence factor genes of ETEC are isolated at the Animal Disease using organisms cultured from infected plasmid borne, the presence of atypical Research and Diagnostic tissue is that cultured organisms may not combinations of genes in ETEC strains is Laboratory at South Dakota State always express fimbriae. In vitro expression not surprising. The low incidence of isola- 1 University, April 2001 to April 2002 is particularly a problem with 987P and tion of each atypical combination suggests F18ab fimbriae.4,21 In addition, heavily that the extra genes add little if any survival Gfenotype No. o encapsulated organisms, most frequently advantage. strains observed with K99 strains, may yield false- K288; LT; STb 12 negative ELISA results.21 References – refereed F618; STa; STb; Stx2e 5 1. Wilson RA, Francis DH. Fimbriae and enterotox- Genotypic analysis by multiplex poly- F518; STa; STb 5 ins associated with E coli serogroups isolated from merase chain reaction (PCR) identifies clinical cases of porcine colibacillosis. Am J Vet Res. All K88+ E9TEC 17 genes for virulence factors, including 1986;47:213–217. + All F18 E0TEC 15 fimbriae K88, K99, 987P, F18, and F41 2. Casey TA, Nagy B, Moon HW. Pathogenicity of All K99+ E8TEC 2 22 porcine enterotoxigenic Escherichia coli that do not (Figure 4). It also identifies genes for tox- express K88, K99, F41, or 987P adhesins. Am J Vet + 2 All F41 ETEC 25 ins LT, STa, STb, and Stx2e. Testing by Res. 1992;53:1488–1492. All 987P+ E8TEC PCR circumvents difficulties associated 3. Westerman RB, Mills KW, Phillips RM, Fortner A7ll other ETEC 3 with expression of genes under laboratory GW, Greenwood JM. Predominance of the ac variant in K88-positive Escherichia coli isolates from conditions and cumbersome assays re- swine. J Clin Microbiol. 1988;26:149–150. 1 Ages of pigs from which the strains quired for identification of enterotoxins. 4. Imberechts H, Bertschinger HU, Nagy B, Deprez were isolated are unknown. However, the multiplex PCR test is not P, Pohl P. Fimbrial colonization factors F18ab and 2 All except three strains had genes of without its own pitfalls. Results are subject F18 ac of Escherichia coli isolated from pigs with postweaning diarrhea and edema disease. Adv Exper another fimbria to interpretation. Superfluous bands may Med Biol. 1997;412:175–183. co-migrate with those representing genes of 5. Francis DH, Grange PA, Zeman DH, Baker DR, virulence factors. (D. Francis, unpublished Sun R, Erickson AK. Expression of mucin-type Methods of diagnosis and data, 2002). Presence of a gene or gene glycoprotein K88 receptors strongly correlates with piglet susceptibility to K88+ enterotoxigenic Escheri- characterization of ETEC fragment evidenced by a band on the agar- chia coli, but adhesion of this bacterium to brush At necropsy, pigs infected with ETEC may ose gel does not necessarily indicate that borders does not. Infect Immun. 1998;66:4050– exhibit no observable lesions other than the gene is expressed as a functional viru- 4055. those associated with dehydration. How- lence factor. Several state-supported veteri- 6. Choi C, Cho W-S, Chung H-K, Jung T, Kim J, Chae C. Prevalence of the enteroaggregative Escheri- ever, some may exhibit mild to moderate nary diagnostic laboratories perform multi- 19 chia coli heat-stable enterotoxin 1 (EAST1) gene in intestinal hyperemia. Histologic exami- plex PCR tests for the virulence genes of isolates in weaned pigs with diarrhea and/or edema nation of the small intestine reveals bacilli diarrheogenic E coli from pigs. disease. Vet Microbiol. 2001;81:65–71. adherent multifocally or diffusely on the 7. Clugston RE, Nielsen NO, Smith DLT. Experi- villous surface (Figure 2). Gram staining of Relative prevalence of E coli strains exhibit- mental edema disease of swine (E.coli enterotox- ing various genotypes in specimens submit- emia). III Pathology and pathogenesis. Can J Comp impression smears from the ileum of most Med. 1974;38:34–43. ted to the South Dakota State University clinically infected pigs reveals large num- 8. Kurtz HJ, Bergeland ME, Barnes DM. Pathologic bers of gram-negative bacilli, typically with Animal Disease Research and Diagnostic changes in edema disease of swine. Am J Vet Res. few other organisms present20 and large Laboratory from April 2001 to April 2002 1969;30:791–806. numbers of E coli can be cultured from is shown in Table 2. More than half of all 9. Runnels PL, Moon HW, Schneider RA. Develop- ETEC strains isolated from pigs with diar- ment of resistance with host age to adhesion of those tissues. Enterotoxigenic E coli cul- K99+ Escherichia coli to isolated intestinal epithelial tured from infected neonatal pigs may be rhea displayed one of three gene patterns: cells. Infect Immun. 1980;28:298–300. either hemolytic or non-hemolytic, but K88, LT, and STb; F18, STa, STb, and 10. Dean EA. Comparison of receptors for 987P pili only hemolytic ETEC colonize weaned Stx2e; or F18, STa, and STb. Most strains of enterotoxigenic Escherichia coli in the small intes- 1,4 not displaying one of these patterns either tines of neonatal and older pigs. Infect Immun. pigs. 1990;58:4030–4035. lacked one or more of the genes or had ad- Confirmation that E coli isolates are patho- 11. Erickson AK, Willgohs JA, McFarland SY, ditional genes for virulence factors. When Benfield DA, Francis DH. Identification of two genic strains may be by phenotypic or ge- multiple E coli isolates were tested from a porcine brush border glycoproteins that bind the notypic analysis. Phenotypic analysis is best single herd outbreak, it was not uncom- K88ac adhesin of Escherichia coli and correlation of performed by assessment for production of these binding glycoproteins with the adhesive por- mon for some isolates to display a typical cine phenotype. Infect Immun. 1992;60:983–988. adhesive fimbriae, either by enzyme-linked virulence gene pattern, while others were 12. Al-Majali AM, Robinson JP, Asem EK, Lamar immunosorbent assay (ELISA) or by indi- missing one of the genes. The clustering of C, Freeman MJ, Saeed AM. Age-dependent varia- rect immunofluorescence assay (IFA).21 isolates missing genes with isolates having a tion in the density and affinity of Escherichia coli Monoclonal antibodies for each type of heat-stable enterotoxin receptors in mice. Adv Exp full complement of genes may suggest that Med Biol. 1999;473:137–145. fimbria are available for these tests. The loss of virulence genes is not uncommon in 13. Grange PA, Erickson AK, Levery SB, Francis ELISA test requires culture of the organ- ETEC. Whether that loss occurs during DH. Identification of an intestinal neutral isms from infected tissues. The IFA may be infection or during laboratory culture is glycosphingolipid as a phenotype-specific receptor performed either on impression smears or for the K88ad fimbrial adhesin of Escherichia coli. unknown. Genes contained within bacte- Infect Immun. 1999;67:165–172. frozen sections of infected intestine or on rial plasmids may readily be disseminated 174 Journal of Swine Health and Production —July and August, 2002 14. Nagy B, Casey TA, Whipp SC, Moon HW. 19. Moxley RA, Duhamel GE. Comparative patho- Susceptibility of porcine intestine to pilus-mediated genesis of bacterial enteric diseases of swine. Adv Exp adhesion by some isolates of piliated enterotoxigenic Med Biol. 1999;473:83–101. Escherichia coli increases with age. Infect Immun. 20. Francis DH. Use of immunofluorescence, gram 1992;60:1285–1294. staining, histologic examination, and seroagglutina- 15. Baker DR, Billey LO, Francis DH. Distribution tion in the diagnosis of porcine colibacillosis. Am J of K88 Escherichia coli-adhesive and nonadhesive Vet Res. 1983;44:1884–1888. phenotypes among pigs of four breeds. Vet 21. Mullaney CD, Francis DH, Willgohs JA. Com- Microbiol. 1997;54:123–132. parison of seroagglutination, ELISA, and indirect 16. Peelman LJ. Genetic investigation of the resis- fluorescent antibody staining for the detection of tance mechanisms of the pig against diarrhea caused K99, K88, and 987P pilus antigens of Escherichia by E. coli. Verhandelingen-Koninklijke Academie voor coli. J Vet Diagn Invest. 1991;3:115–118. Geneeskunde Belgie. 1999;61:489–515. 17. Meijerink E, Fries R, Vogeli P, Masabandi J, References – non refereed Eigger G, Stricker C, Neuenschwander S, 22. Casey TA, Bosworth BT. Diagnosis of E. coli in Bertschinger HU, Stranzinger G. Two alpha (1,2) swine using multiplex PCR. 97th Gen Meet Amer Soc fucosyltransferase genes on porcine chromosome Microbiol. Miami, Florida. 1997;116. 6q11 are closely linked to the blood group inhibitor (S) and Escherichia coli F18 receptor (ECF18) loci. Mamm Genome. 1997;8:736–741. 18. Meijerink E, Neuenschwander S, Fries R, Dinter A, Bertschinger HU, Stranzinger G, Vogeli P. A DNA polymorphism influencing alpha (1,2) fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithe- lium to Escherichia coli F18 adhesion. Immunogenet- ics 2000;52:129–136.

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