Effective genetic markers for identifying the Escherichia coli F4ac receptor status of pigs.

The F4ac receptor locus (F4acR), which encodes susceptibility or resistance to Escherichia coli diarrhoea, is inherited as an autosomal recessive monogenetic trait. F4acR is localized on pig chromosome 13 (SSC13q41-q44) near the MUC13 gene. Two flanking markers (CHCF1 and ALGA0106330) with a high linkage disequilibrium (LD) with F4acR were found to be effective for the genetic identification of F4ac-resistant pigs in the Swiss Large White breed (one recombinant out of 2034 genotyped pigs). Three recombinant boars, one each from the Duroc, Swiss Landrace and Piétrain breeds, were genotyped with seven different markers and phenotyped by means of a microscopic adhesion test. Only ALGA0072075, CHCF1 and CHCF3 indicated the correct phenotype. To test the effect of the resistance allele on production traits, 530 Large White pigs from the national test station were investigated. A significant difference existed among the F4acR locus genotypes in the intramuscular fat content of the longissimus dorsi muscle, whereas no other production traits were influenced by the resistance allele. The frequency of the CHCF1-C and ALGA0106330-A alleles associated with resistance in the Swiss Large White population was 60%, which is advantageous for implementing this trait in a breeding programme to select for E. coli F4ac-resistant animals. The selection of resistant pigs should start on the male side due to the inability of resistant sows to produce sufficient amounts of protecting antibodies in the colostrum. Selection of genetically F4ac-resistant pigs is a sustainable and suitable alternative to decreasing animal loss and antibiotic use due to diarrhoea.

Refined localization of the Escherichia coli F4ab/F4ac receptor locus on pig chromosome 13.

Diarrhoea in newborn and weaned pigs caused by enterotoxigenic Escherichia coli (ETEC) expressing F4 fimbriae leads to considerable losses in pig production. In this study, we refined the mapping of the receptor locus for ETEC F4ab/F4ac adhesion (F4bcR) by joint analysis of Nordic and Swiss data. A total of 236 pigs from a Nordic experimental herd, 331 pigs from a Swiss experimental herd and 143 pigs from the Swiss performing station were used for linkage analysis. Genotyping data of six known microsatellite markers, two newly developed markers (MUC4gt and HSA125gt) and an intronic SNP in MUC4 (MUC4-8227) were used to create the linkage map. The region for F4bcR was refined to the interval SW207-S0075 on pig chromosome 13. The most probable position of F4bcR was in the SW207-MUC4 region. The order of six markers was supported by physical mapping on the BAC fingerprint contig from the Wellcome Trust Sanger Institute. Thus, the region for F4bcR could be reduced from 26 to 14 Mb.

Reduced intestinal colonisation with F18-positive enterotoxigenic Escherichia coli in weaned pigs fed chicken egg antibody against the fimbriae.

Newly weaned pigs were fed a basal diet containing either egg antibody against fimbriae F18 at a high or low level, control egg powder or no egg, and challenged with enterotoxigenic Escherichia coli with fimbriae F18. The challenge was repeated after termination of the antibody treatment. Antibody-containing egg powder was produced by vaccination of hens with semi-purified fimbriae of the two variants F18ab and F18ac. Pigs eating egg powder with antibody against the same fimbrial variant were fully protected, even if the vaccine for the hens was produced with a different serotype devoid of enterotoxins. The effect was dose-dependent. The high dose of antibody against the heterologous variant of fimbriae F18 reduced colonisation at a level which was not significant. Ingestion of egg antibody partially suppressed the build-up of anti-colonisation immunity. Oral application of egg antibodies offers a promising approach for the prevention of infectious diseases of the digestive tract.

Role of fimbriae F18 for actively acquired immunity against porcine enterotoxigenic Escherichia coli.

Enterotoxigenic (ETEC) and enterotoxaemic (ETEEC) Escherichia (E.) coli that express F18 (F107) fimbriate colonize the small intestine and cause diarrhoea and/or oedema disease in weaned pigs. So far, two antigenic variants of F18 can be distinguished with a common antigenic factor designated 'a' and two specific factors called 'b' and 'c'. In this study the existence of crosswise anti-colonization immunity between E. coli strains that express F18ab or F18ac fimbrial variants, respectively, was demonstrated. Weaned pigs of susceptible genotype with respect to susceptibility to adhesion of E. coli with fimbriae F18 were inoculated with E. coli strains 3064STM (0157:K-:H-:F18ab; resistant to streptomycin) and 8199RIF (0141ab:K-:H4:F18ac; resistant to rifampicin). The faecal shedding was compared subsequent to immunization and homologous or heterologous challenge. An enzyme-linked immunosorbent assay (ELISA) was applied to measure IgA, IgM and IgG antibodies against the F18ab and F18ac antigens in saliva, faeces, serum and intestinal wash samples. About 8 log CFU/g of the inoculated strains were found in faeces of all pigs following immunization as well as in non-immunized controls after challenge. Bacterial counts of the inoculated strains after challenge were between 2 and 5 log lower, without any difference between homologous and heterologous challenge. Intestinal colonization with fimbriated E. coli resulted in production of significantly increased levels of anti-fimbrial antibodies, especially IgA, in serum and intestinal wash samples. There were higher levels of homologous than of heterologous anti-fimbrial antibodies. Production of antibodies against F18a or against another common fimbrial antigen is probably responsible for crosswise anti-colonization immunity between E. coli strains with F18ab and F18ac fimbrial variants. Serum F18-specific IgA may be a useful indicator of a mucosal immune response directed against F18 fimbriae.

Inheritance of resistance to oedema disease in the pig: experiments with an Escherichia coli strain expressing fimbriae 107.

Inheritance of resistance to intestinal colonization with E. coli causing oedema disease is hypothesized to be under the control of one locus consisting of two alleles with susceptibility (S) dominating resistance (s). This mode of inheritance was investigated by mating pigs, resistant and susceptible to the disease, and examining the offspring. Weaned piglets were repeatedly inoculated orally with 5 x 10(5) CFU per pig per day of a streptomycin resistant strain of E. coli serotype O139:K12(B):H1:F(107) and susceptibility determined by daily semiquantitative cultural examination of rectal swabs. Using results obtained from offspring, 5 boars were retrospectively assigned the genotype ss, 1 was assigned Ss, and 2 were assigned SS. Nine sows were designated ss, 8 classified Ss and 4 SS. Ninety two pigs resulted from matings regarded as ss x ss; 89 (97%) of these were resistant to colonization and oedema disease. Of the 168 pigs from Ss x ss matings, 83 (49%) were resistant, while only 13 (9%) of 146 pigs from matings with at least one SS parent were classified resistant. The results are compatible with inheritance being controlled by one locus and with susceptibility dominating resistance to oedema disease.

Active oral immunization of suckling piglets to prevent colonization after weaning by enterotoxigenic Escherichia coli with fimbriae F18.

Immunoprophylaxis of porcine oedema disease and post-weaning diarrhoea caused by strains of Escherichia coli expressing fimbriae F18 is an unsolved problem. The study was designed to examine whether vaccination with a live F18ac vaccine of unweaned pigs born to sows with F18ac antibody in the colostrum requires preformed fimbriae in the vaccine, and whether protection against the heterologous fimbrial variant F18ab is induced as well. Genetically susceptible pigs were vaccinated orally on three consecutive days, beginning 10 days before weaning with 10(11) CFU of an F18ac culture. Challenge with a dose of 10(7) CFU of E. coli F18 on three consecutive days was initiated 9 or 11 days after weaning. Eighteen pigs given the fimbriated F18ac vaccine and challenged with a strain of the homologous fimbrial variant were protected against colonization; mean faecal viable counts of the challenge strain were >3 log10 lower than those from the 17 non-vaccinated control pigs. The vaccinated pigs developed a significant rise of F18ac IgA serum antibodies. The 23 pigs which had received the non-fimbriated vaccine showed no significant protection and exhibited much lower serum F18ac IgA ELISA reactivities. Eighteen pigs vaccinated with the fimbriated F18ac and challenged with an F18ab strain had faecal viable counts nearly as high as those from 16 non-vaccinated control pigs. It is concluded that only oral vaccines having preformed fimbriae induce protection limited to the homologous fimbrial variant.

Adhesive fimbriae produced in vivo by Escherichia coli O139:K12(B):H1 associated with enterotoxaemia in pigs.

Two strains of E. coli O139:K12 (B):H1 were compared in vitro and in the intestinal environment. Both strains colonized the small intestines of experimentally inoculated pigs and exhibited in vivo a similar relationship to the microvillus border as enterotoxigenic E. coli (ETEC). Strain 107/86 grown on blood agar expressed numerous long flexible non-haemagglutinating fimbriae which were antigenically distinct from the known fimbriae of porcine ETEC. It adhered in vitro to porcine enterocyte brush border fragments. Strain 124/76 grown on blood agar was devoid of fimbriae and did not adhere to brush border fragments. However, fimbriae morphologically and antigenically indistinguishable from those of strain 107/86 were detected in the intestinal environment by direct immunofluorescence and by immuno electron microscopy.

Designations F18ab and F18ac for the related fimbrial types F107, 2134P and 8813 of Escherichia coli isolated from porcine postweaning diarrhoea and from oedema disease.

The relatedness of the fimbriae produced by eight E. coli strains including type strains with F107 fimbriae, 2134P pili and colonization factor 8813 (preliminary F18), was examined. These strains had been isolated principally from pigs which were affected with postweaning diarrhoea or with oedema disease. The fimbriae were analyzed by means of electron microscopy, slide agglutination, immunofluorescence, immunogold labelling, immuno-diffusion, immunoelectrophoresis and western blot, molecular genetic techniques, and in vitro adhesion. The fimbriae of all the strains were long flexible filaments with a diameter not larger than 4.6 nm showing a zig-zag pattern. Results obtained by the serological techniques confirmed that the fimbriae possessed a common antigenic determinant designated 'a' in addition to a variant-specific determinant designated 'b' or 'c'. Immunoelectron microscopy demonstrated that the determinants 'a' and 'b' or 'a' and 'c' were localized along the same fimbrium. In immunoelectrophoresis, fimbrial extracts of selected strains yielded a single precipitation line towards the cathode. One single major subunit of approximately 15 kDa was recognised in western blots by antisera against the common antigenic determinant and the variant specific determinants. All strains possessed sequences related to gene fedA, coding for the major subunit of fimbriae F107. Two types of fedA-related subunit genes were differentiated, corresponding to the 'ab' and 'ac' types of fimbriae as defined by serological methods. The results demonstrated that F107 fimbriae, 2134P pili and colonization factor 8813 are related, and that two serological variants can be distinguished. We propose designations F18ab (for F107), and F18ac (for 2134P and 8813) in analogy to the nomenclature of F4 fimbriae.

Prevalence of F107 fimbriae on Escherichia coli isolated from pigs with oedema disease or postweaning diarrhoea.

The study comprises fifty 4 to 12 weeks old pigs that died from oedema disease or severe diarrhoea. Smears were prepared from the mucosa of duodenum, jejunum and ileum, and by immunofluorescence F107 fimbrial antigens were detected. E. coli strains were isolated from the intestines and were characterised by slide agglutination (serogroup and F107 fimbriae production), by their cytotoxicity for Vero cells, and by gene amplification (genes coding for the major F107 subunit FedA, the toxin causing oedema disease SLT-IIv, and enterotoxins LTI, STIa and STII). F107 fimbriae were demonstrated in association with E. coli of serogroups O139:K12 and O141:K85a,b but not of serogroup O149:K91:F4a,c. Expression in culture of F107 fimbriae by some isolates gave additional evidence for production of these fimbriae by ETEC strains. The genetic determinant of SLT-Ilv was found in association with F107, and could not be detected in serogroup O149:K91:F4a,c. Gene fedA was demonstrated in two isolates which were devoid of SLT-IIv. Most isolates from cases of oedema disease belonged to serogroup O139:K12 and did not contain enterotoxin genes. Isolates from pigs that suffered from diarrhoea were serotyped O141:K85a,b or O149:K91:F4a,c, and carried at least two enterotoxin genes in their genomes. In a small proportion of the cases F107 antigens were demonstrated in intestinal smears although gene fedA was not detected in the corresponding isolates. The results confirm the importance of F107 fimbriae as virulence factor in oedema disease E. coli strains, but also demonstrate that F107 fimbriae can be found in association with postweaning diarrhoea isolates. In these latter strains enterotoxins were always demonstrated, irrespective of the presence of toxin SLT-IIv.

Fimbrial colonisation factors F18ab and F18ac of Escherichia coli isolated from pigs with postweaning diarrhea and edema disease.

During the last 5 years at least four new types of colonisation factors have been described in association with porcine postweaning diarrhea and edema disease strains of E. coli. Recently, evidence was presented that these fimbrial factors are closely related to each other, and therefore the common denomination F18 was proposed. Until now, two variants F18ab and F18ac were identified that can be distinguished by serology. Alternatively, to circumvent elaborate growth conditions for the optimal expression of F18 fimbriae in vitro, PCR and subsequent restriction enzyme digestion of the amplification product can be used to differentiate F18ab from F18ac positive isolates. Reports that studied the prevalence of F18 positive E. coli show that this factor is present in about 30% to more than 50% of the PWD or ED strains negative for F4, F5, F6 or F41. Susceptibility of pigs to colonisation depends on the availability of intestinal receptors, and is under the control of a chromosomal locus. In young pigs susceptibility increases with age. Intestinal infection with F18 positive E. coli induces protection against repeated colonisation with E. coli bearing the homologous or the heterologous fimbrial variant of F18. Finally, preliminary passive protection studies suggest that F18 antibodies inhibit the colonisation of the pig's intestine by F18ab and F18ac positive strains.

Quantitative detection of antibodies to Mycoplasma suipneumoniae in pigs' sera by an ezyme-linked immunosorbent assay.

A new method for quantitative detection of antibodies to Mycoplasma suipneumoniae in pigs' sera has been developed by the application of an enzyme-linked immunosorbent assay (ELISA). The results show that the ELISA is specific and highly sensitive. In experimentally infected pigs antibodies could be detected several weeks before the clinical manifestation of enzootic pneumonia.

[Main virulence factors in Escherichia coli isolates from swine over two weeks old with edema disease and/or E. coli diarrhea]. / Hauptvirulenzfaktoren bei Escherichia coli-Isolaten von über zwei Wochen alten Schweinen mit Odemkrankheit und/oder Colidiarrhöe.

The OK antigens and the fimbriae F4 of E. coli with haemolysis isolated from 113 cases of oedema disease and/or diarrhoea were identified serologically. The genes for F18 and for enterotoxins LT, STIa and STII as well as Shigatoxin Stx2e were determined by PCR. Fimbrial variants F18ab and F18ac were distinguished by means of indirect immunofluorescence on smears prepared from the intestinal mucosa and from cultures grown under appropriate conditions. Adhesive fimbriae were detected with every case or isolate, respectively, by means of at least one out of the techniques mentioned above. The serogroup O149:K91 with fimbriae F4ac (K88ac) and genes for the enterotoxins LT and STII was most prevalent. Serogroup O139:K12 with fimbriae F18ab and the gene for Stx2e was second, whereas serogroups O141ab and O141ac with fimbriae F18ac and genes for Stx2e, STII and often LT were much less prevalent. The serogroup O147:K89 with fimbriae F18ac, and genes for STIa and STII was detected for the first time in Switzerland.

[Epidemiology of trichophytosis in fattening calves in regard to the prevention of leather defects]. / Epidemiologie der Trichophytie beim Mastkalb im Hinblick auf die Verhütung von Lederschäden.

During the year 1989 12,520 veal calves were visually examined for ringworm at the Zürich abattoir. The mean prevalence of ringworm amounted to 7.7% with a maximum of 12.8% in July and a minimum of 5.1% in March. Epidemiological data were collected at repeated visits from 73 calf fattening farms. Batches of calves were significantly more frequently affected in continuous management systems (51%) than in all-in all-out systems (28%). Ringworm was more prevalent in farms with large groups of calves, and where calves were shorn. Prevention of leather defects necessitates prophylactic measures. The latter are determined by the importance of animal to animal contacts for transmission of the fungus. The health status of calves at purchase and the choice of the management system are therefore of primary importance. Vaccination may be considered in problem herds. A premium for high quality skins might further stimulate the interest of the producers in leather quality.

[Lowering of the incidence of puerperal mastitis in the sow by protection of the mammae from contamination]. / Senkung der Inzidenz von puerperaler Mastitis bei der Sau durch Schutz des Gesäuges vor Verschmutzung.

Twelve sows each farrowed in an experimental pen designed especially for this study or in a crate. Viable counts of enterobacteriaceae were performed in samples taken from the laying area and from the teats. Secretion from every mammary complex was examined repeatedly for bacteria and for somatic cells. The sows in the experimental pen did not lay down in their own faeces. The viable counts in samples from the laying area and the teats were much lower than with the sows kept in farrowing crates. Infection with E. coli was observed in 3 mammary complexes of the sows in the experimental pen as compared to 27 complexes of the sows in the crate. More than half of the infections was detected in the samples taken before farrowing began. In the average the bacteria persisted for 1.3 days. On the first 4 days of life piglets sucking teats with cytologically defined mastitis had an average daily gain of 105 g as compared to 125 g with piglets sucking healthy teats. In conclusion puerperal mastitis is a consequence of faecal contamination of the mammary gland. Soiling of the laying area with faeces and urine can be reduced by improvements in the farrowing accommodations.

[A molecular test for the detection of E. coli F18 receptors: a breakthrough in the struggle against edema disease and post-weaning diarrhea in swine]. / Ein molekularer Test für den Nachweis des E.-coli-F18-Rezeptors: ein Durchbruch im Kampf gegen Oedemkrankheit und Absetzdurchfall beim Schwein.

Oedema disease and post-weaning diarrhoea in swine are associated with the colonization of the intestine with toxigenic Escherichia (E.) coli bacteria of various serotypes. Colonization depends on specific binding between adhesive fimbriae and receptors on the enterocytes. The demonstration of these receptors allows the identification of susceptible and resistant pigs. Direct sequencing of the alpha (1,2) fucosyltransferase gene (FUT1) in swine being either susceptible or resistant to adhesion by F18 fimbriated E.coli revealed a mutation at basepair 307 (M307). Analysis of the mutation in Swiss Landrace and Large White families showed close linkage with the locus controlling resistance and susceptibility to E.coli F18 adhesion (ECF18R). The FUT1 (M307) mutation is a good marker for selection of E.coli of F18 adhesion resistant animals. The mutation is found with variable frequencies in Duroc, Hampshire and Pietrain pigs as well.

[Diarrheal diseases in 1 to 4 week old suckling piglets from problem herds: microbial spectrum, histology, enzyme histochemistry]. / Durchfallerkrankungen bei 1 bis 4 Wochen alten Saugferkeln aus Problembetrieben: Erregerspektrum, Histologie, Enzymhistochemie.

Postmortem investigation was performed on 54 scouring (soon after onset of diarrhea) and 23 healthy suckling piglets. The animals were 1 to 4 weeks of age and were collected from 27 herds. In 21 herds diarrhea had been observed for a long time in piglets in this age group. Coccidia were found in 34 piglets, rotavirus in 2, cryptosporidia in 2, enterotoxigenic E. coli in 6, attaching and effacing E. coli in 1, Cl. perfringens type C in 1, Campylobacter coli in 53 and C. hyointestinalis in 8 piglets. Some of these microorganisms were isolated from healthy piglets as well. TGE- and PED-Virus antigen, as well as Salmonella were not recovered from any of the pigs. Results of the histopathological and enzyme histochemical investigations were in agreement with data from the literature. They confirmed and emphasized the role of coccidia as enteropathogenic agents in the animals examined. On the other hand evidence was shown, that C. coli is non-pathogenic. The role of C. hyointestinalis could not be fully elucidated. At least one sort of enteropathogenic agent was detected in about 68% (n = 37) of the scouring pigs or was present in 81% (n = 17) of the herds, respectively.

Inheritance of porcine receptors for enterotoxigenic Escherichia coli with fimbriae F4ad and their relation to other F4 receptors.

Enteric Escherichia coli infections are a highly relevant cause of disease and death in young pigs. Breeding genetically resistant pigs is an economical and sustainable method of prevention. Resistant pigs are protected against colonization of the intestine through the absence of receptors for the bacterial fimbriae, which mediate adhesion to the intestinal surface. The present work aimed at elucidation of the mode of inheritance of the F4ad receptor which according to former investigations appeared quite confusing. Intestines of 489 pigs of an experimental herd were examined by a microscopic adhesion test modified in such a manner that four small intestinal sites instead of one were tested for adhesion of the fimbrial variant F4ad. Segregation analysis revealed that the mixed inheritance model explained our data best. The heritability of the F4ad phenotype was estimated to be 0.7±0.1. There are no relations to the strong receptors for variants F4ab and F4ac. Targeted matings allowed the discrimination between two F4ad receptors, that is, a fully adhesive receptor (F4adRFA) expressed on all enterocytes and at all small intestinal sites, and a partially adhesive receptor (F4adRPA) variably expressed at different sites and often leading to partial bacterial adhesion. In pigs with both F4ad receptors, the F4adRPA receptor is masked by the F4adRFA. The hypothesis that F4adRFA must be encoded by at least two complementary or epistatic dominant genes is supported by the Hardy-Weinberg equilibrium statistics. The F4adRPA receptor is inherited as a monogenetic dominant trait. A comparable partially adhesive receptor for variant F4ab (F4abRPA) was also observed but the limited data did not allow a prediction of the mode of inheritance. Pigs were therefore classified into one of eight receptor phenotypes: A1 (F4abRFA/F4acR+/F4adRFA); A2 (F4abRFA/F4acR+/F4adRPA); B (F4abRFA/F4acR+/F4adR-); C1 (F4abRPA/F4acR-/F4adRFA); C2 (F4abRPA/F4acR-/F4adRPA); D1 (F4abR-/F4acR-/F4adRFA); D2 (F4abR-/F4acR-/F4adRPA); E (F4abR-/F4acR-/F4adR-).