Protection of rabbits against enteropathogenic Escherichia coli (EPEC) using an intimin null mutant

Abstract

Background: Diarrhea and mortality resulting from infections with enteropathogenic Escherichia coli (EPEC) are of major economic importance in the rabbit meat industry. There is a growing need for an effective vaccine to cope with these problems and to reduce the use of antibiotics. EPEC are characterized by an attaching and effacing virulence mechanism. This is partly mediated by the intimate binding between an adhesin, called intimin, and a translocated receptor (Tir) of prokaryote origin. We constructed an intimin deletion mutant of the rabbit EPEC (REPEC) wild-type strain 97/241.6 (bio-/serogroup 3-/O15) and examined its protective capacity.

Results: After verifying its complete loss of virulence, we used the attenuated strain in vaccination-challenge experiments in which complete protection against a homologous, but virulent, strain was observed. The attenuated strain was able to persist in the intestinal lumen, where it elicited an immune response against EPEC-related virulence proteins, as was shown using an EspB-specific ELISA. Despite the priming of an immune response and the generation of specific antibodies, the intimin mutant was not able to fully protect rabbits against challenges with REPEC strains of other bio-/serogroups.

Conclusion: These data indicate that protection against REPEC infections is at least partly bio-/serogroup dependent and a multivalent vaccine may be needed for protection against the full range of REPEC types. Such a combination vaccine may be developed using intimin null mutants, as the latter were clearly shown to be safe and effective against homologous infections.

Figure 1

PCR amplification of part of the eae gene comprising the deleted 248 bp fragment, using primers 248 FOR and 248 REV. The amplification product of the mutant strain 97/241.6Δeae is shown in lane 2, and that of the wild-type 97/241.6 strain in lane 3. A 100 bp GeneRuler (Fermentas) was used as a size-marker (lane 1).

Figure 2

Immunoblot of the total protein fraction of strain 97/241.6Δeae (lane 2) and wild-type strain 97/241.6 (lane 3). A biotinylated broad-range standard (lane 1) was used as a marker (BioRad). The intimin band (~97 kDa), marked with an arrow, is clearly present in the wild-type strain, but absent in the deletion mutant.

Figure 3

In vitro attachment of fluorescent E. coli strains to isolated intestinal villi. The 97/241.6Δeae REPEC (A) do adhere, but only in small numbers. The insert shows three mutant bacterial cells attached to the enterocytes. DH5α strains (B) do not adhere to the intestinal villi, while B10 strains (C) are strongly adherent. The insert shows multiple E. coli (intense green) attached to the brush border (faint green).

Figure 4

Vaccination experiment followed by challenge with a homologous strain after seven days. Average feed intake of the different groups of rabbits plotted against number of days after challenge. One group was vaccinated with strain 97/246.1Δeae seven days before challenge (day -7; i.e. immediately after weaning), while both the vaccinated and the non-vaccinated group were challenged (day 0) with REPEC 97/223.10 wild-type strain. Significant differences between the non-vaccinated and the other groups are indicated with an asterisk (*:P < 0.05).

Figure 5

Vaccination experiment followed by challenge with a homologous strain after 28 days. Average feed intake of the different groups of rabbits plotted against day after vaccination. Vaccination with strain 97/246.1Δeae was performed immediately after weaning. Both the vaccinated and non-vaccinated groups were challenged at day 28 after vaccination (arrow) with strain 97/223.10. Significant differences between the non-vaccinated and the other groups are indicated with asterisks (***: P < 0.001; **:P < 0.01; *:P < 0.05).

Figure 6

A semi-quantitative assessment of the shedding of the vaccine strain (before day 28) and wild-type strain (after day 28). The day of infection is indicated by the arrow. Grey bars represent the shedding by the vaccinated animals, black bars the shedding by the non-vaccinated rabbits (0 = no shedding, 4 = confluent colonies on agar plate).

Figure 7

Vaccination experiment followed by challenge with heterologous strains after 28 days. Average feed intake of the different groups of rabbits plotted against day after vaccination. Vaccination with strain 97/246.1Δeae (3-/O15) took place immediately after weaning. All groups, with the exception of the control group, were challenged at day 28 after vaccination (arrow) with a virulent strain of the bio-/serogroup indicated in the legend.

Figure 8

A: Detection of the EspB-specific serum antibodies of non-vaccinated and vaccinated rabbits as a function of time. Groups of 12 rabbits were used. The means were significantly different (P = 0.01) from day 14 onward. B: SDS-PAGE followed by silver-staining of a molecular weight standard (1) and purified recombinant EspB (2). The EspB band of approximately 40 kDa is clearly visible.