Comparison of clinical and immunological findings in gnotobiotic piglets infected with Escherichia coli O104:H4 outbreak strain and EHEC O157:H7

Gnotobiotic piglets were derived from four German Landrace sows by Caesarian section in three consecutive trials as described previously [15]. During the trial, sterility was tested by regular bacteriological examination of swabs from the animals and the isolators at the end of the experiment.

In total, 55 piglets were delivered from the four sows. For this study, data of 13 piglets belonging to three different experimental groups were included into evaluation: control group (n = 3) and infection groups E. coli O104:H4 (n = 6) and E. coli O157:H7 (n = 4), respectively. All other piglets were of low vitality, did not stay sterile or had been used for other experiments.

Piglets were infected orally 12 h after birth with 5 ml bacterial suspension using a curved olive headed probe outreaching the root of tongue. The control group received sterile sodium chloride (0.9% NaCl) (Table 1).

Escherichia coli O157:H7 strain 86-24 originates from a meat associated EHEC outbreak in Walla Walla WA [18]. This intimin positive strain has an A/E phenotype, synthesizes Stx2 and is able to cause a D + HUS like disease in gnotobiotic piglets [15].

Escherichia coli O104:H4 strain e2975 is a stool isolate obtained from a 16-year-old male patient, who was admitted to the University Hospital Essen, Essen, Germany with bloody diarrhoea during the 2011 EHEC outbreak in Germany. In the further course the patient developed HUS and also exhibited seizures. The strain was geno-serotyped using the E. coli PanType AS-2 DNA oligonucleotide microarray [19] from Alere Technologies (Jena, Germany). Stx subtyping was performed with the ShigaToxType AS-2 from Alere Technologies [20]. Furthermore, the complete genome of the strain was determined on an Illumina HiSeq sequencing system (Illumina, San Diego, CA, USA). Like all isolates of that outbreak, it bears the serotype O104:H4 and is a chimera between EHEC and EAEC. It displays a blended phenotype with characteristics of both E. coli pathotypes [7, 21, 22]. The strain produces Stx2 variant a, lacks intimin and enterohemolysin, is positive for iha encoding an adherence-conferring protein homologous to IrgA [23] and shows an ESBL resistance pattern due to plasmid encoded TEM-1 and CTX-M-M15 β-lactamases. It also possesses mchB coding for the microcin H47 activity protein [24].

Escherichia coli cultures for oral infection were prepared in advance and stored in 0.9% NaCl at room temperature. Briefly, 4 ml overnight culture of the respective bacterial strain were grown in LB broth (LBB; 10 g/l tryptone, 10 g/l NaCl, 5 g/l yeast extract) and inoculated at an optical density OD₆₀₀ of 0.06 in 100 ml LBB the following day. When a specific OD₆₀₀ was reached, individually determined for each strain according to growth curves, bacteria were harvested by centrifugation (50 ml tubes, 4000×g at 4 °C for 10 min). The supernatant was discarded and the pellet was washed once with phosphate buffered saline (PBS; 1.4 mM NaCl, 116 mM NaH₂PO₄, 18 mM KH₂PO₄) to remove any free Stx or degradation products. Cultures were diluted in 0.9% NaCl and colony forming units (CFU) were determined by plating and counting serial dilutions. Bacterial suspensions were stored at room temperature until further use. Piglets were orally infected with 5 ml liquid culture with doses ranging from 2.08 × 10⁸ to 2.20 × 10⁸ CFU for E. coli O157:H7 and 2.19 × 10⁸ to 1.00 × 10⁹ CFU for the E. coli O104:H4 outbreak strain.

A visual inspection of animals was performed every 2 h and clinical scores were recorded every 4 h. Appetite, neurological signs and status of hydration were separately assessed as physiological (score 0), mild (score 1) and severe (2) and scores were added to calculate a total score. A total clinical score for each individual was generated by dividing sum of all scores through number of examination time points.

If clinical scores were assessed to be ≥2 at two consecutive examination times the respective pig was euthanised due to animal welfare reasons. The experiment was finalised on days 9–12 after infection by euthanasia of piglets (Table 1). Immediately after death, pigs were removed from the isolator, weighed and dissected under a S2 laminar air flow workbench.

Red and white blood cells as well as clinical chemistry parameters were determined according to routine diagnostic methods described elsewhere with slight modifications [25]. Acute phase proteins haptoglobin (Tridelta Phase Haptoglobin Assay, Tridelta Development Limited, Maynooth, Ireland), C-reactive protein (Phase Porcine CRP Assay, Tridelta Development Limited) and lipopolysaccharide-binding proteins (LBP ELISA, Hycult Biotech, Uden, Netherlands) were determined according to the manufacturer’s instructions of used detection kits.

Urine was collected on day 6 and day of death and processed for routine diagnostics, including refractometry, urine chemistry and testing by Combur 9 test strips (Roche Diagnostics, Mannheim, Germany). In sediments the amount of bacteria, leukocytes, superficial squamous epithelial cells, round epithelial cells and deeper transitional epithelial cells as well as crystals were assessed by microscopical inspection. The glomerular filtration rate (GFR) and the fractional excretions (FE) of water and Na were calculated based on the endogenous marker creatinine, which is exclusively eliminated by glomerular filtration [26].

Faecal samples were obtained on day 4, 6 and day of necropsy. A 1:10 starting solution was prepared by dissolving 100 mg faeces in 900 µl PBS and was then diluted further up to 10⁻⁸. 10 µl of each serial dilution were spotted clockwise on Müller-Hinton agar (Oxoid, Thermo Fisher Scientific, Wesel, Germany), sorbitol MacConkey agar (SMAC, Oxoid) and CHROMagar ESBL (MAST DIAGNOSTICA, Reinfeld, Germany) in parallel to obtain a general overview of the bacterial load in the fecal samples. For detailed analysis 50 µl of bacterial suspension were spread on an entire agar plate.

All tissue samples were fixed and stained according to routine histological methods as described previously [27]. After immediate fixation in 10% buffered formalin samples were embedded in paraffin wax. Paraffin sections (6 µm) were either stained with hematoxylin–eosin (HE, hemalaun after Delafield) or with Masson´s trichrome stain for routine histological examination.

Multiple small 1 mm³ tissue blocks from the renal cortex, the colonic mucosa, and the cerebellum were sampled for electron microscopy and fixed in 5% glutaraldehyde in phosphate-buffered saline (PBS). After an adequate fixation time of at least 24 h samples were washed in PBS, postfixed for 90 min in 1% osmium tetroxide in double-distilled water, subsequently rinsed in 30, 50 and 70% ethanol. In a next step tissue was infiltrated with uranylacetate and phosphoric tungstic acid within 2 h. After subsequent 60-min-treatments with 90% ethanol, 100% ethanol and propylene oxid, tissue was stored in epon (Epon 812, Fluka, Buchs, Switzerland): propylene oxid in a 1:1 mixture at 4 °C overnight. The next day samples were infiltrated with pure epon for 1 h and poured in silicon forms, followed by two 24-h-polymerization steps at 35 and 45 °C and one 60-h-polymerization step at 60 °C. Toluidine blue stained thin sections (1.5 μm) from each tissue block were inspected for selection of areas for EM ultrathin sectioning. Ultrathin sections were cut on a Leica Ultramicrotome (Leica Ultracut S, Vienna, Austria) and stained with uranyl acetate (Sigma-Aldrich, Vienna, Austria) and lead citrate (Merck, Darmstadt, Germany). Ultrathin sections were examined with a Zeiss TEM 900 electron microscope (Carl Zeiss, Oberkochen, Germany) operated at 50 kV.

In accordance with a previously established protocol [28], PBMCs were isolated from heparinized blood by density gradient centrifugation using lymphocyte separation medium (Pancoll human, density 1.077 g/ml, PAN Biotech, Aidenbach, Germany), which had been adjusted by PBS (without Ca²⁺ and Mg²⁺, PAN Biotech) dilution to a density of 1.075 g/ml. Mesenterial and ileocaecal lymph nodes were dissected during necropsy from surrounding tissue, pooled for each individual animal and further processed as described elsewhere [29].

Isolated cells from blood and lymph nodes were suspended in PBS supplemented with 10% porcine plasma (in-house preparation). Cell labelling was performed in 96-well round-bottom microtiter plates (Greiner Bio One, Frickenhausen, Germany) with 2 to 5 × 10⁵ cells per sample. Antibodies and second-step reagents used for labelling are listed in Table 2. Prior to use, optimal working dilutions for all antibodies and second-step reagents had been determined in titration experiments. Mastermixes of antibodies were prepared freshly before use. After addition of primary antibodies samples were incubated for 20 min at 4 °C, followed by two washing steps in 200 μl of PBS. A plate shaker was used for resuspension of cells after washing steps. Secondary reagents were added and also incubated for 20 min at 4 °C, followed by two washings steps with PBS + 10% porcine plasma. For discrimination of dead cells Fixable Near-IR Dead Cell Stain Kit (Invitrogen, Carlsbad, CA, USA) was used during the second incubation according to manufacturer’s instructions. In a further incubation step fixation and permeabilization was performed with the BD Cytofix/Cytoperm kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer`s instructions. This allowed the staining of the intracellular epitope recognized by the anti-CD79α antibody (Table 2).

For each fluorochrome single stain samples were prepared at the time when the staining protocol was established and used for automatic calculation of compensation by FACSDiva software Version 6.1.3 (BD Biosciences).

Samples were analysed on a BD FACSCanto II flow cytometer equipped with a high-throughput sampler and three lasers (405, 488 and 633 nm, BD Biosciences) and subsequently analysed by FACSDiva software (Fig. 1). In PBMC numbers of analysed cells ranged from 12,000 to 132,000 and in lymph nodes cells from 14,000 and 72,000. Total numbers of cell subpopulations were calculated based on white blood cell counts and percentages of cells resulting from flow cytometry.

The following circulating cytokines/chemokines were determined by applying FMIA on serum samples as described elsewhere [30]: interleukin (IL)-1-beta (IL1β), IL4, IL8, IL10, IL12, interferon gamma (IFNγ) and chemokine ligand (CCL) 2. Used antibodies and standards are listed in Table 3.

For statistical analysis laboratory diagnostic findings from samples taken between day 4 and 6 after infection were compared between the groups by Mann–Whitney U test (SPSS Statistics for Windows, Version 20.0, Armonk, NY: IBM Corp.). Differences were assessed to be significant at p ≤ 0.05. Information about results always refers to this time range, unless otherwise stated. Results obtained after day 6 were not statistically evaluated due to small sample numbers.

While animals in the control group stayed healthy, all animals infected with E. coli O104:H4 showed a transient reduction of milk intake. Four pigs out of this group became dehydrated due to diarrhoea starting at day 4 after infection. Two animals developed mild neurological disorders between days 2–6 after infection. Three pigs infected with E. coli O157:H7 developed severe clinical signs and had to be euthanized on days 4 and 5 according to preassigned termination criteria. In two of these pigs, neurological disorders were determined, which were characterised by swaying, incoordination, head shaking and excitation. The third animal was severely dehydrated due to liquid diarrhoea. One animal infected with E. coli O157:H7 survived until the end of trial (day 11 p.i.) but was continuously dehydrated due to diarrhoea. In infected animals, the mean average daily weight gain (ADWG) was 98 g (E. coli O104:H4 infected animals) and 74 g (E. coli O157:H7). ADWG of E. coli O157:H7 infected animals was significantly lower than the ADWG in control animals (115 g, p = 0.034). During necropsy liquid intestinal content was found in the gut of all infected animals. Clinical data are summarized in Table 1.

No differences between groups regarding haematocrit, haemoglobin levels as well as erythrocyte and thrombocyte numbers were found (Table 4). A higher percentage of normoblasts was found early after infection in E. coli O157:H7 infected animals in comparison to control pigs (p = 0.032) and E. coli O104:H4 infected pigs (p = 0.010). One animal infected with E. coli O157:H7 showed thrombocytopenia on day 5 after infection (215 × 10⁹ thrombocytes/l).

Electrolyte and protein concentrations as well as enzyme activities did not differ between the groups. Haptoglobin was significantly increased in E. coli O157:H7 infected pigs compared to negative controls (p = 0.032). CRP and LBP concentrations in infected animals were not significantly increased (Table 4).

Specific weights of urine and other urine parameters did not differ between the groups. All infected piglets showed viable bacteria in the native urine sediments. A selection of results is presented in Table 5.

Escherichia coli O157:H7 was reisolated in all infected animals in similar amounts in pure culture. E. coli O104:H4 was reisolated in four animals at all sampling times. In two pigs out of this group (ID 6 and 7) reisolation failed on the day of death. Faeces of all animals within the control group were sterile as expected. In all faeces samples from infected animals Shiga toxin ELISA gave clearly positive results. Findings resulting from bacteriological examinations and ELISA are summarized in Table 1.

Thrombotic microangiopathy in the kidneys was found in two pigs infected with E. coli O157:H7 (Fig. 2) and three pigs infected with E. coli O104:H4. By electron microscopy in a pig infected with E. coli O157:H7, dilation of the subendothelial space within the glomerula with the functional consequence of leakage was seen (Fig. 3d). One animal infected with E. coli 157:H7 showed acute spongiosis, another animal moderate focal liquefaction necrosis in the brainstem (Fig. 4). Histological findings from CNS, kidney and C. ascendens are summarized in Table 6. Results of examination of tissue samples from other organs are not shown, although minor inflammatory alterations in individual piglets were found. Electron microscopy revealed differences in the attachment of the bacteria. While in E. coli O157:H7 infected pigs prominent A/E lesions were found in enterocytes with pedestal formation and deprivation of microvilli (Fig. 3a, c), E. coli O104:H4 stayed in the mucus layer in distance to the epithelium (Fig. 3b).

In blood, total number of leukocytes decreased in E. coli O157:H7 infected animals compared to the control group (p = 0.046) and E. coli O104:H4 infected animals (p = 0.020). Two animals infected with E. coli O157:H7 developed leukocytopenia (3.1 and 3.4 G/l, respectively; reference range 3.9–19.7 G/l; [25]). One animal infected with E. coli O104:H4 developed leukocytosis (20.5 G/l).

All pigs infected with E. coli O157:H7 showed low numbers of polymorphonuclear neutrophils (PMN) beneath lower reference value (0.07–1.27; reference range 1.89–11.27 G/l, [25]), one animal infected with E. coli O104:H4 developed granulocytosis (14.56 G/l) (Fig. 5a).

For the absolute numbers of total T and B cells present in blood in tendency a decrease was found in E. coli infected animals compared to control animals and this decrease was more pronounced in E. coli O157:H7 infected pigs; somehow reflecting the drop of leukocytes counts described above (Fig. 5b, c, respectively). However, within the pool of mesenterial and ileocaecal lymph nodes no obvious change of the percentages of these two lymphocyte subsets was found between the different groups of animals. For CD172 α⁺ cells (consisting mainly of monocytes after removal of granulocytes by density gradient centrifugation) in blood very heterogeneous numbers were found across all groups (Fig. 5d). In the lymph nodes, percentages of these CD172 α⁺ myeloid cells were significantly decreased in E. coli O157:H7 (p = 0.034) and E. coli O104:H4 infected animals (p = 0.039) in comparison to control group, respectively.

Next to T and B cells NK cells were addressed as another major lymphocyte subset, including NKp46-defined subsets which have been investigated in pigs previously [31, 32]. Absolute numbers of NK cells and NKp46⁺ NK cells in blood were slightly enhanced in E. coli O157:H7 infected animals and even more in E. coli O104:H4 infected animals (Fig. 5e, g, respectively). No such changes were found for NKp46⁻ NK cells in blood (Fig. 5f) and no obvious changes were found in NK cells and related NK-cell subsets in lymph nodes (Fig. 5e–g).

In addition to total T cells (CD3⁺) the subpopulation of CD4⁺ T cells (CD3⁺CD4⁺ phenotype; representing putative T-helper cells) was investigated (Fig. 5h). Similar to the absolute number of total T cells (Fig. 5b) CD4⁺ T cells were reduced in the blood of infection groups (not significant). In tendency, a slight increase of the percentages of this population was found in the lymph nodes of E. coli O157:H7 infected animals (Fig. 5h). A peculiarity of porcine CD4⁺ T cells is the up-regulation of CD8α molecules on the cell surface following activation [33]. Hence, within one of the staining panels of our flow cytometry analyses monoclonal antibodies against CD8α were included (Table 2), allowing the investigation of CD8α expression in CD4⁺ T cells. However, frequencies of CD8α⁺ CD4⁺ T cells were extremely low (data not shown), which is in accordance with previously published data of newborn piglets [28]. No obvious changes in absolute counts in blood or frequencies within lymph nodes was found for this very rare T cell subpopulation between infected and control pigs (data not shown). Within the T cell subpopulation of CD3⁺CD4⁻, CD8α⁺ cells (representing cytolytic T cells) were analysed (Fig. 5i). In blood of E. coli O157:H7 infected animals total numbers of cytolytic T cells were decreased compared to control pigs and E. coli O104:H4 infected animals. But these differences were not significant. In lymph nodes, percentage of this population was significantly decreased in E. coli O157:H7 infected animals compared to animals infected with E.coli O104:H4 (p = 0.011). γδ T cells are another prominent T cell subpopulation in the blood [34] and secondary lymphatic organs of pigs [35]. For absolute numbers of γδ T cells in blood, a substantial variation among different piglets was found but without an obvious link to E. coli infection (Fig. 5j). Within lymph nodes percentages of γδ T cells were slightly, but not significantly reduced in infection groups. Expression of the cell surface molecules CD2, CD8α and CD27 could be recently related to different stages of γδ T cell development. For CD2 it was shown that already in the thymus two different lineages of γδ T cells can be identified by this marker [36]. For CD8α and CD27 published data suggest that within CD2⁺ γδ T cells a CD8α⁺CD27⁻ phenotype is related to a late stage of differentiation [35]. Hence, we analysed expression of CD2, CD8α and CD27 in γδ T cells also in this study (Fig. 5k–m). Absolute numbers in blood for CD2⁻ γδ T cells were slightly increased in E. coli-infected groups (Fig. 5k), whereas CD2⁺ γδ T cells were slightly reduced (Fig. 5l). No clear infection-related changes were found in the CD2-defined γδ T cell subsets analysed in lymph nodes (Fig. 5j, k). When CD2⁺ γδ T cells were analysed for CD8α and CD27 expression, the vast majority of cells had a CD8α⁺CD27⁺ phenotype (Fig. 5l, m). The absolute numbers of this γδ T cell subset in blood showed no infection-related changes, whereas the percentages of CD8α⁺CD27⁺ γδ T cells within CD2⁺ γδ T cells in lymph nodes was slightly enhanced in infection groups.

Cytokine/chemokine responses are summarised in Table 7. IFNγ was significantly increased in both infection groups compared to healthy controls (p = 0.038 in E. coli O104:H4 and p = 0.031 in E. coli O157:H7 infected animals, respectively). IL8 in measurable amounts (34.2 pg/ml) could only be found in a single animal infected with E. coli O157:H7 on day 11 after infection (data not shown). IL1β could be measured at very low and comparable levels in all groups. No IL4 response could be measured in any animal (data not shown). IL12 levels were comparably low in all animals, with the exemption of one animal in each infection group (714.l8 pg/ml in an E. coli O104:H4 infected animal and 6247 pg/ml in an E. coli O157:H7 infected animal, respectively). A decrease in CCL2 could be assessed in E. coli O157:H7 infected animals in comparison to the control group (p = 0.034). IL10 was significantly increased in piglets infected with E. coli O104:H4 (p = 0.020) and not significantly increased in E. coli O157:H7 infected animals.