David H. Francis, PhD
This diagnostic note has not been peer refereed.
Department of Veterinary Science, South Dakota State University; Brookings, South Dakota 57007-1396
Francis DH. Colibacillosis in pigs and its diagnosis. Swine Health Prod. 1999;7(5):241-244.
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Colibacillosis is a major cause of illness and death in young pigs. The condition is caused by enterotoxigenic and other strains of Escherichia coli. Characteristics of the causative agents and the animals at risk are discussed. Methods for diagnosis of the condition are given.
Colibacillosis is a major cause of illness and death in neonatal and recently weaned pigs. The disease is usually caused by enterotoxigenic strains of the bacterium Escherichia coli, although nonenterotoxigenic strains of that organism may also occasionally cause the disease. Diarrhea is typically fluid and profuse, and frequently results in severe dehydration and circulatory shock. Enterotoxins produced by the enterotoxigenic E. coli (ETEC) strains pathogenic to pigs include heat-labile enterotoxin (LT), and/or heat-stable enterotoxins STa (STI) or STb (STII). These organisms also produce fimbrial adhesins that mediate the adherence of the bacterium to the mucosal surface. The fimbriae produced include K88 (F4), K99 (F5), 987P (F6), F41, and F18 (F107 and 2134P). Although less common, some strains produce a Shiga toxin (Stx2e) and may cause edema disease in addition to colibacillosis. Also uncommon are strains that produce no toxins, but efface the microvilli of the epithelial cells to which they attach (Helie P, et al. Proc Int Congr Vet Soc. 1990).1 Such strains contain eae genes, which have been associated with attachment/effacement. Porcine attaching-effacing E. coli strains are very similar to those that cause diarrhea in human infants, and are known as enteropathogenic E. coli (EPEC). Because many strains of E. coli isolated from animals are nonpathogenic, it is important to identify the virulence factors produced by ETEC or EPEC strains, or the genes that encode those factors, to establish the etiology of diarrhea.
Inherent susceptibility or resistance of pigs to ETEC appears to be a function of age and/or genetic background. Resistance of pigs to E. coli-expressing K99 or 987P arises with age. Age-associated resistance develops gradually and becomes more-or-less complete by 2 weeks of piglet life.2-4 Interestingly, age-acquired resistance of calves to K99+ E. coli occurs very rapidly and is complete by 48 hours of age.5 Pigs are resistant to infection by F18+ E. coli at birth, but become susceptible after several weeks of life.6 Inheritable resistance to colibacillosis caused by K88+ and F18+ ETEC is well documented, but has not been reported with regard to E. coli which produces other adhesive fimbriae.7-9 Inherent resistance to, attachment of, and susceptibility to K88+ and F18+ E. coli are autosomal recessive traits. Resistance is achieved by failure to produce the receptor to which the fimbriae adhere on epithelial brush border membranes.9 Enterotoxigenic E. coli-expressing K88 and F18 account for essentially all postweaning colibacillosis in pigs.2,10,11 K88+ E. coli is believed to be responsible for a majority of neonatal colibacillosis cases as well.12 Recent studies suggest that about 50% of pigs in common breeds inherit resistance to K88+ organisms.13 Thus it appears that pigs inherently susceptible to K88+ ETEC account for a disproportionately high proportion (at least 75%) of all colibacillosis. Therefore, selective breeding for resistance to K88+ and perhaps F18+ E. coli could have a significant economic impact on the swine industry.
The technology for identifying inherently resistant animals is currently quite primitive and cumbersome, yet testing for and selecting such animals may be economically justified. Currently, methods of susceptibility/resistance phenotype analysis require either laboratory analysis of intestinal biopsy specimens or specimens from progeny at slaughter. Two laboratory tests for phenotype analysis are available. The most definitive of these tests employs a Western blot protocol.14 This test requires larger specimens than can be collected by biopsy, expensive reagents, and considerable technical expertise.
Although it can be less specific, the other test--a brush border/bacteria aggregation test--requires only small specimens, no expensive reagents, and minimal training to perform. However, some laboratory equipment, including a centrifuge and a microscope with a phase-contrast condenser, is required. K88+ bacteria are incubated with whole enterocytes or osmotically prepared enterocyte brush-border vesicles obtained from the pig in question. Bacteria/brush border suspensions are viewed by phase contrast microscopy for the adherence of bacteria to brush borders (Figure 1). Adherence of numerous bacteria to brush borders is highly correlated with piglet susceptibility. Specimens from pigs < 6 weeks of age may give false-positive results.9 Sows of the resistant phenotype should not be mated with boars of the susceptible phenotype or boars that remain uncharacterized because such sows may be unable to protect susceptible offspring from K88+ ETEC (susceptibility is dominant over resistance). Sows of the resistant phenotype do not produce anti-K88 antibody subsequent to oral exposure with K88+ ETEC or K88 antigen.15 However, they probably produce circulating (IgG) anti-K88 antibody following parenteral vaccination.
Colibacillosis is a disease of the small intestine, and the condition of that organ must be assessed in diagnosing the disease. Strains of some serotypes of ETEC colonize the entire small intestine, while strains of other serotypes colonize only the distal portion of the small intestine. For that reason, specimens examined by the clinician or submitted to a laboratory for examination should include distal ileum. High concentrations of E. coli in pure or nearly pure culture in the ileum are indicative of colibacillosis.
Simple clinical methods
The concentration of E. coli in the intestines can easily be estimated by preparing and examining Gram-stained impression smears of the mucosal surface of the small intestine (Figure 2). More than 100 bacteria per 1000x microscopic field is indicative of colibacillosis.17 Alternatively, one may estimate the E. coli concentration by culture of the mucosa of the ileum on blood and/or a differential medium agar such as Tergitol-7 or MacConkey. Abundant growth of E. coli of a single colony type is indicative of colibacillosis.
In addition to the above mentioned methods for estimating the concentration of E. coli in the intestine, determining whether such organisms adhere to epithelial cell brush borders is useful in diagnosing colibacillosis. Only E. coli that are capable of adherence are considered significant in the disease. Adherent bacteria, if present, can be observed by examining Hematoxylin-eosin (H & E)-stained or Wright-Giemsa-stained ileal tissue sections (Figure 3), or unstained wet mount preparations of mucosal scrapings.16 The wet mounts should be examined using phase-contrast microscopy. Perhaps one of the better approaches to determining the existence of ETEC infection is by examining immunofluorescence- or immunohistochemical-stained ileal impression smears or histologic sections for bacteria expressing adhesive fimbriae (Figure 4).16 Examination of anti-fimbriae antibody-stained intestinal sections reveals the fimbriae, if any are expressed by the E. coli, provides an estimate of the concentration of the organisms, and shows whether they adhere to the epithelium. Rabbit antisera or monoclonal antibodies specific for the various adhesive fimbriae are used in immunofluorescent or immunohistochemical tests. However, a limitation of this approach is that only E. coli expressing the fimbriae to which the antibodies are directed will be detected. Cases involving attaching-effacing E. coli or E. coli-expressing uncharacterized adhesive fimbriae will be missed. The ideal specimen for diagnosing colibacillosis is a pig euthanized during acute disease and subjected to necropsy immediately after euthanasia. Because the mucosal epithelium of the small intestine is subject to rapid autolysis and postmortem colonization by colonic bacteria, use of animals that have succumbed to diarrhea is discouraged in attempting diagnosis.
Identifying markers of virulence
In many cases it is desirable to characterize the E. coli isolate obtained from an animal to provide evidence that the isolate is virulent. Characteristics that suggest virulence include serogroup, adhesive fimbriae, and exotoxins. A serologic approach to characterization may be pursued. Serogrouping and fimbriae testing should include analysis for the serogroup and fimbrial antigens (Table 1). The serologic approach to E. coli isolate characterization is relatively simple to accomplish, and reagents and equipment are not expensive. This approach has weaknesses in that some (albeit few) E. coli strains diarrheogenic in pigs do not belong to a defined set of serogroups.3 In addition, the expression of some fimbriae is subject to phase variation or growth conditions difficult to duplicate in the laboratory.6,17,18 Thus, test sensitivities are limited, and false-negative results may occur relatively frequently.
Another approach to the characterization of E. coli strains isolated from diarrheic pigs is genetic. One may test for presence of DNA sequences consistent with those that encode virulence determinants of interest. DNA-based tests could include assays for the genes of adhesive fimbriae, enterotoxins, Stxe, and eae. An example of a DNA-based test for genes of E. coli virulence determinants is the multiplex polymerase chain reaction (PCR) test recently developed at the National Animal Disease Center in Ames, Iowa (Bosworth BT, et al. 97th Gen Meet Amer Soc Microbiol. 1997; 116), which is now available in some diagnostic laboratories (Figure 5). While gene-based tests are technically more demanding than serologic tests, they overcome the problems associated with poor expression of some virulence determinants under in vitro conditions. However, these tests do not determine whether a gene is actually encoding a specific virulence factor, only whether a specific segment of DNA is present in the E. coli strain being tested. DNA segments from genes containing mutations that render them functionally inactive, or that are similar in sequence but different in function, can lead to false positive results.
1. Janke, BH, Francis DH, Collins JE, Libal MC, Zeman DH, Johnson, DD. Attaching and effacing E. coli infections in calves, pigs, lambs, and dogs. J Vet Diag Invest. 1989; 1:6-11.
2. Wilson RA, Francis DH. Fimbriae and enterotoxins associated with E. coli serotypes isolated from clinical cases of porcine colibacillosis. Am J Vet Res. 1986; 47:213-217.
3. Runnels PL, Moon HW, Schneider RA. Development of resistance with host age to adhesion of K99+ Escherichia coli to isolated intestinal epithelial cells. Infect Immun. 1980; 28:298-300.
4. Dean EA. Age-specific colonization of porcine intestinal epithelium by 987P-piliated enterotoxigenic Escherichia coli. Infect Immun. 1989; 57:82-87.
5. Smith HW, Halls S. Observations by the ligated intestinal segment and oral inoculation methods on Escherichia coli infections in pigs, calves, lambs and rabbits. J Pathol Bacteriol. 1967; 93:499-529.
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7. Bertschinger HU, Stamm M, Vogeli P. Inheritance of resistance to oedema disease in the pig: Experiments with an Escherichia coli strain expressing fimbriae F107. Vet Microbiol. 1993; 35:79-89.
8. Rutter JM, Burrows MR, Sellwood R, Gibbons RA. A genetic basis for resistance to enteric disease caused by E. coli. Nature (London). 1975; 257:135-136.
9. Francis DH, Grange PA, Zeman DH, Baker DR, Sun R, Erickson AK. Expression of mucin-type glycoprotein K88 receptors strongly correlates with piglet susceptibility to K88+ enterotoxigenic Escherichia coli, but adhesion of this bacterium to brush borders does not. Infect Immun. 1998; 66:4050-4055.
10. Wittig W, Klie H, Gallien P, Lehmann S, Timm M, Tschape H. Prevalence of the fimbrial antigens F18 and K88 and of enterotoxins and verotoxins among Escherichia coli isolated from weaned pigs. Zentralbl-Bakteriol. 1995; 283:95-104.
11. Hide EJ, Connaughton ID, Driesen SJ, Hasse D, Monckton RP, Simmons NG. The prevalence of F107 fimbriae and their association with Shiga-like toxin II in Escherichia coli strains from weaned Australian pigs. Vet. Microbiol. 1995; 47:235-243.
12. Moon HW, Bunn TO. Vaccines for preventing enterotoxigenic Escherichia coli infections in farm animals. Vaccine. 1993; 11:213-220.
13. Baker DR, Billey LO, Francis DH. Distribution of K88 Escherichia coli-adhesive and nonadhesive phenotypes among pigs of four breeds. Vet. Microbiol. 1997; 54:123-132.
14. Erickson AK, Willgohs JA, McFarland SY, Benfield DA, Francis DH. Identification of two porcine brush border glycoproteins that bind the K88ac adhesin of Escherichia coli and correlation of these binding glycoproteins with the adhesive porcine phenotype. Infect Immun. 1992; 60:983-988.
15. Van den Broeck W, Cox E, Goddeeris BM. Receptor-dependent immune responses in pigs after oral immunization with F4 fimbriae. Infect Immun. 1999; 67:520-526.
16. Francis DH. Use of immunofluorescence, gram's staining, histologic examination, and seroagglutination in the diagnosis of porcine colibacillosis. Am J Vet Res. 1983; 44: 1884-1888.
17. Mullaney CD, Francis DH, Willgohs JA. Comparison of seroagglutination, ELISA, and indirect fluorescent antibody staining for the detection of K99, K88, and 987P pilus antigens of Escherichia coli. J Vet Diagn Invest. 1991; 3:115-118.
18. Bosworth BT, Dean-Nystrom EA, Casey TA, Neibergs HL. Differentiation of K88ab+ from F18ac+ Escherichia coli by single-stranded conformational polymorphism analysis of the major fimbrial subunit gene (fedA). Clin Dign Lab Immunol. 1998; 5:299-302.