Differential Virulence of Clinical and Bovine-Biased
Enterohemorrhagic Escherichia coli O157:H7 Genotypes
in Piglet and Dutch Belted Rabbit Models
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Citation
Shringi, S. et al. “Differential Virulence of Clinical and BovineBiased Enterohemorrhagic Escherichia Coli O157:H7 Genotypes
in Piglet and Dutch Belted Rabbit Models.” Infection and
Immunity 80.1 (2011): 369–380. © 2011 American Society for
Microbiology
As Published
http://dx.doi.org/10.1128/iai.05470-11
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American Society for Microbiology
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Author's final manuscript
Accessed
Wed May 25 18:52:58 EDT 2016
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http://hdl.handle.net/1721.1/78007
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1
Differential virulence of clinical and bovine-biased enterohemorrahgic E. coli
2
O157:H7 genotypes in piglet and Dutch Belted rabbit models
3
4
Smriti Shringi1a, Alexis García2a, Kevin K. Lahmers1, Kathleen A. Potter1, Sureshkumar
5
Muthupalani2, Alton G. Swennes2, James G. Fox2, Carolyn J. Hovde3, Douglas R. Call1
6
and Thomas E. Besser1*
7
1
8
Department of Veterinary Microbiology and Pathology, Washington State University,
9
10
Pullman, WA 99164, USA
2
Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge,
11
MA 02139, USA
3
12
Department of Microbiology, Molecular Biology, and Biochemistry, University of
13
Idaho, Moscow, Idaho 83844, USA
a
14
Co-first author
15
16
Running title: Differential virulence of EHEC O157 genotypes
17
18
*
19
Mailing Address: Food and Waterborne Disease Research Program, Veterinary
20
Microbiology and Pathology, College of Veterinary Medicine, Washington State
21
University P.O. Box 647040, Pullman, WA 99164-7040.
22
Phone: 509-335-6075.
23
Fax: 509-335-8529.
Corresponding Author: Thomas E. Besser
1
24
E-mail: tbesser@vetmed.wsu.edu.
25
ABSTRACT:
26
27
Enterohemorrhagic E. coli O157:H7 (EHEC O157) is an important cause of food and
28
waterborne illness in the developed countries. Cattle are a reservoir host of EHEC O157
29
and a major source of human exposure through contaminated meat products. Shiga toxins
30
(Stx) are an important pathogenicity trait of EHEC O157. The insertion sites of the Stx-
31
encoding bacteriophages differentiate EHEC O157 isolates into genogroups commonly
32
isolated from cattle but rarely from sick humans (bovine-biased genotypes, BBG) and
33
commonly isolated from both cattle and human patients (clinical genotypes, CG). Since
34
BBG and CG share the cardinal virulence factors of EHEC O157 and are carried by cattle
35
at similar prevalence, the infrequency of BBG among human disease isolates suggests
36
that they may be less virulent than CG. We compared the virulence potential of human
37
and bovine isolates of CG and BBG in newborn conventional pig and weaned Dutch
38
Belted rabbit models. CG-challenged piglets experienced more severe disease
39
accompanied by earlier and higher mortality than BBG-challenged piglets. Similarly,
40
CG-challenged rabbits were more likely to develop lesions in kidney and intestine when
41
compared with the BBG-challenged rabbits. The CG strains used in this study carried
42
stx2 and produced significantly higher amount of Stx, whereas the BBG strains carried
43
stx2c gene variant only. These results suggest that BBG are less virulent than CG and that
44
this difference in virulence potential is associated with the Stx 2 sub-type(s) carried
45
and/or the amount of Stx produced.
46
2
47
INTRODUCTION:
48
49
Enterohemorrhagic E. coli O157:H7 (EHEC O157) is a major cause of food- and
50
water-borne illnesses characterized by bloody diarrhea, hemorrhagic colitis (HC) and
51
life-threatening hemolytic uremic syndrome (HUS) in developed nations across the globe
52
(27, 38, 43). The lack of specific treatment leads to 2 to 5% mortality among HUS
53
patients. The annual incidence of EHEC O157 infection is estimated to be >70,000 cases
54
in the US (19). The CDC surveillance data shows that the number of EHEC O157
55
outbreaks increased recently from 27 in 2006 to 39 in 2007 (8, 9). In 2008, the CDC
56
reported 1.12 cases of foodborne EHEC O157 infections per 100,000 population in the
57
USA (7). The infectious dose of EHEC O157 is estimated to be very low (10-100
58
bacteria) (54), and transmission occurs primarily via contaminated food, water, or direct
59
contact with infected animals (18, 20, 25, 47). Cattle are considered a major reservoir of
60
EHEC O157 due to the frequent associations of EHEC O157 outbreaks with the
61
consumption of contaminated beef and dairy products, confirmed by molecular
62
epidemiologic evidence of identical strain types (3, 5). Cattle colonized with EHEC O157
63
remain asymptomatic while shedding up to 105 cfu / g of feces typically for periods of
64
days to weeks (51). EHEC O157 is defined as an adulterant when identified in foods, and
65
therefore, in addition to its public health significance, this organism is responsible for
66
significant economic losses to the food industry due to mandatory recalls of contaminated
67
meat and meat products.
68
69
Nearly all EHEC O157 isolates harbor two cardinal virulence factors: production
of one or more antigenically distinct Shiga toxins (Stxs) and the presence of a
3
70
chromosomal pathogenicity island referred to as the Locus of Enterocyte Effacement
71
(LEE) (34, 43). Stxs are encoded in late genes of one or more lambdoid bacteriophages
72
that are integrated into the EHEC O157 bacterial chromosome at specific sites (11, 22,
73
31, 32, 41, 45, 48). Shiga toxins are important virulence factors associated with the
74
systemic effects of EHEC infection including HC and HUS (28, 56). The LEE encodes a
75
type III secretion system (T3SS), including an effector molecule Tir (translocated intimin
76
receptor), and Intimin, an EHEC O157 surface expressed molecule that interacts with Tir
77
and that is also a putative adhesion factor (44). The LEE is required for EHEC O157
78
colonization and formation of attaching and effacing (A/E) lesions on intestinal
79
epithelium (15, 40).
80
Diverse genotyping systems for EHEC O157 strains (4, 29, 30, 36, 41, 52, 62, 63,
81
65) emphasize the role of bacteriophage, including Stx-encoding bacteriophages, in
82
generating genetic diversity in EHEC O157. Several of these methods identified
83
genotypes occurring at different frequencies in cattle and human host (4, 29, 36, 63).
84
Recent studies comparing some of these genotyping methods suggest that they result in
85
generally concordant classifications (33, 60). Using Stx-encoding bacteriophage insertion
86
(SBI) sites as a marker for strain differentiation, EHEC O157 isolates can be classified
87
into two genogroups: bovine-biased genotypes (BBG, predominantly SBI genotypes 5
88
and 6, found primarily in cattle and rarely isolated from sick humans) and clinical
89
genotypes (CG, predominantly SBI genotype 1-3, found in both cattle and humans) (4,
90
52, 61). Both BBG and CG carry and express the cardinal virulence factors of EHEC
91
O157 (4). Despite the similar prevalence of both genogroups in cattle, indicating
92
approximately equal human exposure, the relatively rare occurrence of BBG as a cause of
4
93
human disease suggests that BBG may be less virulent than CG. Therefore, this study
94
was designed to evaluate the virulence potential of human and bovine strains belonging
95
to BBG and CG, using a newborn conventional pig (NCP) and Dutch Belted rabbit
96
(DBR) models. We show that strains of EHEC O157 belonging to BBG are less virulent
97
and cause less severe disease in both animal models. This difference in virulence
98
potential is associated with the presence of different Stx 2 sub-type(s) and/or the quantity
99
of Stx produced by BBG strains.
100
101
MATERIALS AND METHODS
102
103
Piglet husbandry and experimental challenges
104
All animal experiments were conducted according to the protocols approved by the
105
WSU Institutional Animal Care and Use Committee (IACUC). Piglet challenges were
106
conducted as described previously (14) with a few modifications as follows. Piglets were
107
obtained from WSU swine center at ~4 h after birth, and confirmed EHEC O157-free by
108
testing fecal swabs obtained prior to challenge by using immunomagnetic separation
109
(Dynabeads anti-E. coli O157, Invitrogen Dynal AS, Oslo, Norway). Piglets were orally
110
treated with nalidixic acid (25 mg) followed by sodium bicarbonate (10 ml, 10% w/v) 1 h
111
later. Immediately after sodium bicarbonate treatment, piglets were orally challenged
112
with nalidixic acid resistant EHEC O157 (~1010 cfu, NalR). Piglets were fed Enfamil
113
infant milk formula (Mead Johnson & company, IN USA) three times daily beginning at
114
210 ml/day and gradually increasing to 450ml/day on day 6 and 7 post infection (PI).
115
Nalidixic acid (25 mg every 8 h) was orally administered to each piglet throughout the
5
116
experiment. To avoid confounding by potential genetic factors, each piglet cohort (4 – 6
117
piglets from single litter) was randomly allocated equally to CG and BBG strain
118
challenges. Piglets were observed daily for EHEC O157-associated clinical signs (Table
119
1) for up to 7 days PI. Fecal samples from each piglet were collected for quantification of
120
EHEC O157. Piglets were treated with oral fluids and electrolytes (Enterolyte H.E., Oral
121
Powder, Pfizer) and flunixin meglumine (Banamine 1 mg/lb BW IM q24 h) when
122
showing signs of diarrhea and moderate CNS disease (shivering, tremors or mild
123
seizures). Piglets exhibiting severe clinical signs (severe seizures, lateral recumbency, or
124
paresis) were humanely euthanized. At 7 days PI, all surviving piglets were humanely
125
euthanized. Complete necropsies were performed on all piglets, and internal organs were
126
collected and processed for bacteriological, histopathological and electron microscopic
127
examination. All clinical and pathologic assessments were conducted by veterinarians
128
blind to the challenge genotype.
129
130
Bacterial strains for NCP
131
Twenty EHEC O157 strains (Table 2) were randomly selected from the bank of SBI-
132
genotypes of EHEC O157 at Washington State. EHEC O157 strain Sakai (CG-3, kindly
133
provided by Dr. Thomas S. Whittam, NFSTC-MSU) isolated from an outbreak in Japan
134
served as positive control. In order to generate spontaneous nalidixic acid resistant
135
variants (NalR), each EHEC O157 strain was inoculated into brain heart infusion (BHI)
136
broth, incubated at 37°C overnight (~16 h), and 1 ml was plated on Sorbitol MacConkey
137
agar plates (Hardy Diagnostics, Santa Maria, CA, USA) supplemented with nalidixic acid
138
(30 µg/ml) (SMAC-N). For animal challenge studies, NalR strains were inoculated in 200
6
139
ml of trypticase soy broth (TSB) and incubated at 37°C overnight with shaking at 200
140
rpm. Aliquots of these cultures were then stored in glycerol (10% v/v) stocks at -80°C
141
until use.
142
Bacteriological culture for piglet challenge
143
EHEC O157 were detected and enumerated in fecal samples and intestinal tissues of
144
each piglet by decimal dilution plating on SMAC-N agar plates using a spiral plater
145
(Whitley Automated Spiral Platter, Don Whitley Scientific Ltd, UK). Ten sorbitol non-
146
fermenting colonies from each specimen were tested for lactose fermentation
147
(MacConkey’s agar, Becton, Dickinson and Company, Sparks, MD, USA) and for beta-
148
glucuronidase activity (EC-MUG agar, Hardy Diagnostics, Santa Maria, CA, USA).
149
Presumptive EHEC O157 colonies (sorbitol negative, lactose positive and beta-
150
glucuronidase negative) were confirmed by O157 latex agglutination test (E. coli PRO
151
O157, Hardy Diagnostics, USA). One confirmed EHEC O157 colony recovered from
152
each sample was stored (18% buffered glycerol in BHI, -80°C) for subsequent
153
comparisons with their respective challenge strains by SBI-genotyping (61) and by
154
pulsed field gel electrophoresis (PFGE) following XbaI restriction digestion (6, 12).
155
156
Histopathological examination of piglet tissues
157
Duodenum, jejunum, ileum, cecum, spiral colon, distal colon, recto-anal junction (RAJ),
158
kidney and brain collected at necropsy were immediately fixed in 10% neutral buffered
159
formalin, and subsequently paraffin-embedded, sectioned, and stained with hematoxylin
160
and eosin for histopathologic examination. Brain tissue sections were also stained with
161
Luxol-fast blue stain (59). Immunohistochemistry was performed on selected tissues
7
162
using a mouse IgG1 anti-O157 monoclonal antibody and the AEC detection kit
163
(SIGNET, Covance, CA). For EM, tissues were fixed in McDowell’s and Trump’s 4F:1G
164
(37). Histopathologic scoring was based on observed bacterial attachment, necrosis,
165
inflammation, vasculitis, and presence or absence of fibrin. Scoring was performed
166
independently by two ACVP board-certified pathologists (KP and KL) and by SS blinded
167
to the identity of the challenge strain. The histopathological lesions in brain and kidney
168
were scored by two observers (KP and KL), and in intestine by three observers (KP, KL,
169
and SS).
170
171
Rabbit experiments
172
All rabbit experiments were approved by the IACUC at Massachusetts Institute of
173
Technology. Three challenge experiments were performed using weaned 7 to 8-week-old
174
Dutch Belted rabbits (DBR). Each challenge experiment included five rabbits each for
175
positive controls inoculated with EHEC O157 strain EDL933 (CG-3) and four to five
176
rabbits each for negative controls inoculated with PBS, along with six rabbits each
177
inoculated with the CG or BBG challenge strain of EHEC O157. In each experiment,
178
rabbits were fasted overnight and sedated prior to blood collection and orogastric
179
intubation. Experimental and control rabbits were inoculated with 10 ml of 10% sterile
180
sodium bicarbonate to neutralize gastric acidity. Experimental rabbits received 1-2 × 109
181
colony-forming-units of EHEC O157 re-suspended in sterile PBS and control rabbits
182
received sterile PBS (vehicle only). Food and water were provided ad libitum following
183
inoculation. Rabbits were monitored closely for clinical signs and body weight
184
measurements were recorded. Fecal cultures were performed on days 1 and 3 PI. Rabbits
8
185
were euthanized on day 6 or 7 PI; however, rabbits exhibiting severe clinical signs were
186
euthanized before the end of the study (day 4 PI). Prior to euthanasia, blood samples were
187
collected for clinical pathological evaluation. Complete necropsies were performed on all
188
the rabbits and tissue samples from various organs including cecum and kidneys were
189
collected for histopathological evaluation.
190
191
Bacterial strains for DBR
192
Five challenge strains were selected for the rabbit infection studies (Table 2). Stock
193
inocula containing ~ 1010 cfu was prepared and stored in 1 ml aliquots at -80°C until use.
194
195
Bacteriological culture for DBR
196
Challenge strains were detected…
197
198
Histopathological examination of rabbit tissues
199
Tissues were fixed in formalin, embedded in paraffin, sectioned at 5 µm, and stained
200
with hematoxylin and eosin. Kidney sections were also stained with Carstairs’ to detect
201
fibrin deposition. Kidney and intestinal (cecal) sections were evaluated by a board-
202
certified veterinary pathologist (SM) blinded to the identity of the challenge strain.
203
Scoring criteria for histopathological evaluation included renal glomerular (capillary
204
thickening, luminal constriction, fibrin thrombi, white blood cell infiltration, mesangial
205
deposits, changes in bowman’s capsule and space) and vascular (intimal swelling, mural
206
degeneration, perivascular edema, and red blood cell fragmentation) criteria as well as
207
assessment of intestinal vasculopathy (17).
9
208
C-reactive protein analysis
209
The concentration of C-reactive protein (CRP) was measured in serum samples from
210
rabbits in experiment 3 using a commercially available rabbit C-reactive protein ELISA
211
(Immunology Consultants Laboratory, Inc., Newberg, OR) following the manufacture’s
212
directions. Rabbit serum samples were diluted 1:400 in assay diluent buffer for testing.
213
Samples were adjusted to a 1:10,000 dilution and retested if they did not fall within the
214
working range of the assay’s standard curve. The normal range of rabbit CRP is 0-31
215
µg/ml (Gentry PA, 1999).
216
217
stx2 gene typing
218
The stx2 gene variants present in the challenge strains were determined using PCR-
219
RFLP (13). In this method, restriction patterns generated by HaeIII differentiate stx2
220
from stx2c while restriction pattern generated by PvuII differentiate stx2 and stx2c from
221
other stx2 variant types.
222
223
Stx Quantitation
224
Stx production in NalR challenge strains and their nalidixic acid susceptible wild-type
225
parents (WT-NalS) were assessed using ELISA (Premier EHEC test kit, Meridian
226
Bioscience, Cincinnati, OH). Briefly, for each strain, single colonies from blood agar
227
plates were inoculated into 10 ml of TSB, incubated at 37ºC overnight and used to
228
prepare fresh TSB subcultures by inoculating 500 µl of overnight culture into 10 ml of
229
fresh TSB. This subculture was processed for bacterial counts and three aliquots (1 ml
230
each) were transferred into three wells of 96-well culture plates followed by addition of
10
231
1) carbadox (0.5 µg/ml, Sigma-Aldrich, St. Louis, MO), 2) nalidixic acid (30 µg/ml) or 3)
232
no inducing agent. The plates were incubated overnight at 37°C with shaking at 250 rpm.
233
Overnight cultures were diluted 1:100 in sterile TSB immediately followed by 1:2
234
dilution in sample diluent provided in the ELISA kit and 100 µl aliquots of diluted
235
samples were tested by ELISA according to the manufacturer’s protocol.
236
237
Statistical analysis
238
Statistical analyses were performed using NCSS 2007 version 07.1.19 (23). For NCP
239
challenge study, the genogroups were compared using following statistical tests. The
240
clinical scores were analyzed using one- way ANOVA. The five clinical scoring
241
categories (diarrhea, CNS disease, appetite, vomiting and abnormal vocalization) were
242
analyzed using two-way generalized linear method (glm). Survival data was compared
243
using Kaplan-Meier survival analysis and the Log-Rank test. Bacterial load recovered
244
from five segments of intestine (ileum, cecum, spiral colon, distal colon and RAJ) of
245
piglets was compared using two-way glm. Scores for histopathological lesions in
246
intestinal tract were analyzed using two-way (genogroup and pathologist) glm.
247
Association of development of brain lesions in the piglets with the development of CNS
248
disease or with the genogroup of the EHEC O157 strain used to challenge the piglets was
249
assessed using Fisher’s exact test. In the DBR model, statistical analyses were performed
250
using GraphPad Prism. The Mann-Whitney two-tailed test was used to compare the
251
lesion scores of experimental versus control rabbits. All rabbits inoculated with EDL933
252
(positive controls) and those inoculated with PBS (negative controls) in the three
253
challenge experiments were grouped for the statistical analyses. Association between stx
11
254
gene typing and genogroup of the challenge strain was assessed using chi-square test. Stx
255
ELISA OD450 scores were analyzed using three-way (growth condition, nalidixic acid
256
susceptibility, and genogroup) glm. Multiple comparisons were only done when the
257
overall statistical model was significant using Tukey-Kramer test. Each statistical test
258
was conducted at the significance level of P≤0.05.
259
260
261
262
RESULTS
263
264
NCP Model
265
Clinical Signs
266
Piglets were scored for disease using a standard scoring system (Table 1). The piglets
267
cohorts did not differ significantly (P=0.138). Piglets challenged with the clinical
268
genotype strains (CG-1 and CG-3) exhibit higher clinical scores when compared with the
269
piglets challenged with bovine-biased genotype strains (BBG-5 and BBG-6) (P=0.005,
270
Table 3). Piglets challenged with CG strains and BBG strains had clinical scores that did
271
not differ significantly from the positive control Sakai strain and sham inoculated piglets,
272
respectively (P>0.439). In individual genotype comparisons, CG-3 challenged animals
273
had significantly higher clinical scores than BBG-6 inoculated piglets (P=0.023, Fig 1),
274
while CG-1 and BBG-5 challenged piglets showed intermediate clinical scores. Diarrhea,
275
CNS symptoms, appetite, vomiting, and abnormal vocalizations were scored separately.
276
Analyses of these categories suggested that CNS disease scores were significantly higher
12
277
for piglets challenged with CG strains when compared with the piglets challenged with
278
BBG strains (P=0.004, Table 3). No other scoring category differed significantly between
279
CG and BBG challenged piglets.
280
Mortality
281
Challenge with CG strains resulted in earlier and higher mortality than challenge with
282
BBG strains (P=0.002, Fig 2 and Table 3). Sham-inoculated piglets survived until the end
283
of experiment. CG-challenged piglets showed mortality similar to the piglets challenged
284
with positive control strain Sakai (P=0.852). Out of 10 piglets challenged with CG
285
strains only one piglet (challenged with strain E12064) survived until the end of the
286
experiment. On the contrary, out of 10 piglets challenged with BBG strains only two
287
piglets (challenged with strain E12053 and E12156) died on days 2 and 4 PI.
288
Magnitude of bacterial load
289
The mean±SEM log10 cfu/cm2 of EHEC O157 recovered from intestine (ileum, cecum,
290
spiral colon, distal colon and RAJ) were significantly higher for piglets challenged with
291
CG strains, when compared with the piglets challenged with BBG strains (P=0.003,
292
Table 3). The mean±SEM log10 cfu/cm2 of EHEC O157 recovered from ileum, cecum,
293
spiral colon, distal colon and RAJ were 3.86±0.40, 4.92±0.32, 4.56±0.22, 5.00±0.26 and
294
5.14±0.27, respectively. Although the number of bacteria recovered from large intestine
295
was higher when compared with small intestine, statistically significant differences were
296
only observed between RAJ and ileum.
297
Negative control piglets were housed in separate cages within the same room and
298
at the same time as EHEC challenged piglets. EHEC O157 was recovered from some
299
negative control piglets, albeit in small numbers, indicating the likely cross-transmission
13
300
of EHEC O157 between challenged and sham-inoculated animals. Therefore, SBI
301
genotypes and PFGE profiles of challenge strains were compared with the strains
302
recovered from each piglet to determine the level of cross-transmission, if any. In all but
303
two cases, only the challenged strain was recovered from each experimental piglet,
304
indicating that cross-transmission was uncommon. Both of the cross contaminated piglets
305
were challenged with BBG-6 strain. In one of these BBG-6 challenged piglets, one out of
306
6 recovered isolates belonged to a genotype (CG-3) that was different from the genotype
307
of the challenge strain. In other BBG-6 challenged piglet, all seven isolates recovered
308
belonged to non-BBG-6 genotypes (BBG-17, one isolate; BBG-5, one isolate; CG-1, five
309
isolates). However, the clinical scores of these two piglets were lower than the piglets
310
that were challenged with the cross-contaminating CG strains in the same cohort,
311
suggesting that this transmission had minimal effect on the clinical outcomes. Isolates
312
recovered from challenged animals occasionally differed in PFGE profiles by one or two
313
bands from the challenge or contaminating strain. Two genotypes that were not included
314
in the study (genotype 9, negative for all the six PCR fragments in SBI genotyping, and
315
genotype 17, positive for only yehV-left junction PCR fragment in SBI genotyping) were
316
recovered from two piglets. These piglets had been either challenged or contaminated by
317
BBG-5 strain (positive for stx2 gene and yehV-left junction PCR fragments in SBI
318
genotyping) suggesting loss of stx2 encoding bacteriophage or loss of stx2 gene either in-
319
vivo or during the isolation process. However, the PFGE profiles of these recovered
320
isolates were closely related to the challenge or the contaminating BBG-5 strain,
321
confirming their origin.
322
Histopathology
14
323
The histopathological scores for intestinal tract in CG and Sakai challenged piglets were
324
higher than BBG and sham inoculated piglets but did not reach statistical significance
325
(P=0.071, Table 3). However when the two genogroups (CG and BBG) were compared
326
the histopathological scores of intestinal tract for CG-challenged piglets were
327
significantly higher than BBG-challenged piglets (P=0.02). Brain lesions were scored as
328
present or absent (Fig 3). The number of piglets in which brain lesions were observed by
329
at least one observer were 5/10, 2/10, 2/4, and 0/4 in CG, BBG, positive and negative
330
control groups, respectively. The number piglets showing brain lesions did not differ
331
significantly between CG and BBG challenged piglets (P=0.159). However, 7 out of 11
332
piglets that died or were euthanized following severe CNS signs did exhibit brain lesions
333
while none of 9 piglets that did not exhibit severe CNS signs or mortality developed brain
334
lesions (P<0.003). No kidney lesions were observed in any of the piglets.
335
336
DBR Model
337
Three challenge experiments were performed to compare the virulence potential of CG
338
and BBG strains in the DBR model. The most severe clinical signs were observed in
339
rabbits from experiment three. Rabbits in this experiment were challenged with EDL933,
340
E3046 and E5252. Three rabbits (one in each infection group) was euthanized early (at 4
341
days PI) due to the severity of diarrhea. The % change in body weight of these three
342
rabbits infected with EDL933, E3046, and E5252 at day 4 PI was -1.8%, -4.6%, and
343
11.5%, respectively.
344
Pre- and post-inoculation clinical pathological parameters including hematocrit, white
345
blood cell count, percent heterophils, platelet count, blood urea nitrogen, total protein,
15
346
and albumin were compared for rabbits in each group; however, analyses did not reveal
347
consistent changes associated with disease. In experiment three, increased serum
348
concentration of CRP was detected in rabbits that were euthanized on day 4 PI due to
349
severe disease: 451.4 µg/ml (EDL933), 162.1 µg/ml (E3046), and 399.5 µg/ml (E5252).
350
These values were approximately 4-fold higher than the group mean and were consistent
351
with published rabbit values (65-350 µg/ml) indicative of an acute phase response
352
(Gentry PA, 1999).
353
On gross necropsy, infected rabbits exhibited serosal hemorrhages near the cecocolic
354
junction (Fig. 4). Specifically, these hemorrhages were observed in approximately 33%
355
(5/15), 17% (1/6), and 83% (5/6) of rabbits infected with EDL933, E3046, and E5252,
356
respectively. Serosal hemorrhages were not observed in other infected groups or
357
uninfected controls.
358
Significant lesions in the cecum and kidneys of rabbits are summarized in Table 4.
359
Relative to sham-inoculated control rabbits, significant intestinal vasculopathy (Fig.5 B
360
and C) was observed in most O157:H7 infected rabbits except in those infected with
361
strain E5880. .Rabbits infected with E5252 developed the most renal lesions that were
362
significant and included both glomerular and vascular lesions such as glomerular
363
capillary thickening (Fig.5E), luminal constriction (Fig.5F), erythrocyte fragmentation
364
(Fig.5F), intimal swelling and perivascular edema. Rabbits infected with E3046
365
developed significant renal vascular lesions that included intimal swelling and
366
perivascular edema. .
367
368
stx2 gene typing
16
369
All the CG strains tested in this study carried stx2, and two of the five CG-1 strains also
370
carried stx2c. In contrast, all BBG strains carried stx2c alone (P<0.001).
371
372
Stx Quantitation
373
The number of bacteria in the initial subculture used for induction by nalidixic acid or
374
carbadox or left un-induced was ~107cfu/ml (range, 1.6x107cfu/ml to 7.8x107 cfu/ml).
375
There was no significant difference in the bacterial counts (mean±SEM log10 cfu/ml) of
376
BBG (7.54±0.06) when compared with CG (7.65±0.03) at the time of induction
377
(P=0.289). Induction with carbadox resulted in a significant increase in the Stx ELISA
378
OD450 scores irrespective of the genogroup (P=0.05), while induction with nalidixic acid
379
did not result in a significant increase in Stx ELISA OD450 scores by any group, probably
380
due to the NalR trait of all challenge strains. CG strains produced significantly higher Stx
381
ELISA OD450 scores than BBG strains under non-inducing and carbadox inducing
382
conditions (P=0.05). In individual genotype comparisons, challenge strains belonging to
383
CG-1 and CG-3 produced significantly higher Stx ELISA OD450 scores than BBG-5 and
384
BBG-6 under carbadox inducing conditions (P=0.05, Fig. 6). However, under non-
385
inducing conditions, challenge strains belonging to CG-1 produced significantly higher
386
Stx ELISA OD450 scores than BBG-5 and BBG-6. Nalidixic acid induction resulted in no
387
significant difference between genotypes. There was no difference in production of Stx
388
between of NalS and NalR strains within the genotypes under any of the tested growth
389
conditions, suggesting that the use of nalidixic acid to suppress the normal flora of the
390
experimental piglets did not affect in vivo Stx production during these challenge
391
experiments.
17
392
393
DISCUSSION
394
395
The relatively decreased virulence of BBG strains support the hypothesis that not all
396
EHEC O157 SBI genotypes are equally pathogenic. This is evident based on the higher
397
clinical disease severity, earlier and higher mortality, and more severe histopathological
398
lesions observed for piglets challenged with CG strains. Similar results were obtained in
399
rabbits where animals challenged with CG strains exhibited significantly higher
400
histopathological scores. In contrast, the BBG strains generally showed little virulence in
401
either animal model. Our observations are consistent with the previously published study
402
where EHEC strains from healthy cattle were, on average, less virulent in gnotobiotic
403
piglets than EHEC isolated from human disease outbreaks (2). Here we can refine these
404
observations to show that virulence in animal models is associated with specific EHEC
405
O157 genotypes, and that these differences in virulence correlate with the frequency of
406
occurrence of these genotypes among clinical isolates rather than in the animal reservoir.
407
Further, we show that the CG and BBG genotypes tested in these animal models are
408
characterized by consistent differences in their Stx2 variant gene content, with stx2
409
consistently present in the CG strains (with or without stx2c) while stx2c is consistently
410
present and stx2 is consistently absent in the BBG strains.
411
Our piglet model differed in several ways from that of previously published
412
reports (2). In contrast to the gnotobiotic piglet model, in which ~24 h old piglets were
413
inoculated with~109 cfu of EHEC O157, we used ~4 h old nalidixic acid treated
414
conventional piglets that were pretreated with 10% sodium bicarbonate before challenge
18
415
with ~1010 cfu of EHEC O157. The piglet model closely reproduces some aspects of
416
EHEC O157-induced systemic disease of human infection. However, one of the
417
important clinical manifestations of EHEC O157 infection in piglets is CNS disease,
418
which is often associated with brain lesions (2). In this case, the animal model
419
demonstrates lesions in a tissue (brain) typically unaffected in infected humans; however,
420
the brain lesions and CNS disease are arguably analogous to Stx-mediated renal disease
421
in humans as they are the result of Stx being absorbed into circulation, damaging Stx-
422
receptor-expressing endothelial cells resulting in microangiopathy and target tissue
423
damage (14, 56). CG strains induced more severe CNS symptoms (seizures, convulsions,
424
paresis, and lateral recumbency) as compared with BBG strains. In addition, there was a
425
significant association between CNS disease and the presence of histopathological lesions
426
in the brain.
427
Several recent studies have shown that EHEC O157 infected piglets also develop
428
kidney lesions that appear to be associated with Stx production (2, 21, 46). However,
429
kidney lesions were not observed in the piglets infected with either BBG or CG strains in
430
this study. Perhaps due to the rapid onset of severe CNS disease in piglets, the renal
431
disease is only minimally expressed before death (2). Genetic differences (the breed of
432
pigs), the age at the time of infection and time of euthanasia after the appearance of first
433
CNS signs may have contributed to the observed differences in the severity of brain
434
lesions and lack of kidney lesions in our study compared with the previous studies using
435
piglet model.
436
437
We present direct evidence of some cross-transmission in our piglet studies;
however the direction and degree of cross-transmission were not sufficient to confound
19
438
the strong bacterial genotype effects on piglet virulence observed here. Cross-
439
transmission of EHEC O157 from infected to uninfected piglets has been described
440
earlier by Cornick and Vukhac (10), suggesting that EHEC O157 was readily transmitted
441
among swine via contaminated aerosols and suggesting that a very low dose is required
442
for piglet infection. Similarly, minor differences in bacteriophage content and PFGE
443
pattern between challenge strains and fecal isolates as reported here have been reported
444
previously, for example in experimental bovine infections (64). The infrequency of these
445
observations in our study demonstrates the overall stability of genotypes during piglet
446
infections.
447
The DBR model for EHEC O157 exhibits renal lesions that mimic HUS in
448
humans (17). Therefore, we used this model to evaluate the relative virulence of BBG
449
and CG strains and their capacity to induce intestinal and renal lesions. Rabbits
450
experimentally infected with different EHEC O157 genotypes developed a wide range of
451
intestinal and renal histopathological changes suggesting that the virulence of EHEC
452
O157 varies depending on the strain and/or genotype. More specifically, CG-3 strain
453
E5252 appeared to be the most pathogenic based on the extent and severity of the renal
454
and intestinal lesions. Furthermore, the increased serum concentration of CRP in the most
455
clinically affected rabbits suggested that this acute phase protein may represent a
456
potential biomarker of severe EHEC infection and disease. CRP has been considered a
457
predictive factor for hemolytic uremic syndrome development in humans with E. coli
458
O157:H7 infection (Ikeda et al, 2000).
459
460
Several studies have associated the amount of Stx produced and/or type of stx
gene present to the virulence of EHEC O157 strains (2, 16, 39, 42). Therefore, we
20
461
compared the stx2 gene subtype present and the quantity of Stx produced by the EHEC
462
O157 strains used in this study. CG strains carry stx2 alone or in combination with stx1 or
463
stx2c genes while BBG strains carry only stx2c gene. Moreover, under bacteriophage
464
inducing conditions using carbadox, CG strains produced higher Stx ELISA OD450 scores
465
when compared with the BBG strains. These results suggest that the presence of stx2
466
gene and/or production of higher amounts of Stx may be contributing to the increased
467
virulence of CG strains. Although, similar relationship between amount of Stx produced
468
and virulence has been reported earlier (2, 16, 39, 42), the quantification of Stx depends
469
on the specificity of antibody used in the kit, which detects Stx1 or Stx2 and Stx2c with
470
different sensitivity (26, 42). In our study, we observed higher Stx ELISA scores for the
471
CG strains when compared with the BBG strains. However, it was not possible for us to
472
attribute this difference solely to the amounts of toxin produced by these strains as they
473
also differ in the type of stx2 gene content. Therefore, a careful interpretation is needed
474
while using ELISA to compare the amount of Stx produced among EHEC O157 strains.
475
HUS/severe disease in humans have been linked to the strains that carry stx2, stx2c
476
or in combination (1, 42). 9 out of 10 piglets challenged with strains carrying stx2,
477
whether alone or in combination with stx1 or stx2c, died. However, only 2 out of 10
478
piglets challenged with the strains carrying stx2c alone died. These results suggest that
479
the EHEC O157 strains that carry stx2c alone are likely to be less pathogenic when
480
compared with the strains that carry stx2 alone or in combination with stx2c or stx1. The
481
higher amount of Stx produced suggested by higher Stx ELISA OD450 scores of CG
482
strains carrying stx2 gene could explain the severe CNS signs that were observed in CG
483
challenged piglets when compared with the BBG challenged piglets. Other researchers
21
484
have observed the histopathological lesions in the brain and kidney and have attributed
485
these to the systemic effect of Stx (2, 21, 46). In addition to the amount of Stx produced,
486
differences in stx2 gene content could result in differences in the affinity of the toxin to
487
the receptors in the brain and eventual differences in their ability to cause disease or Stx-
488
induced damage to the brain (42).
489
Besides inducing systemic disease, recent studies suggest that Stx2 production in
490
the intestine can also promote colonization, by enhancing the expression of host cell
491
intimin receptors, using invitro cell culture and mouse model (35, 50). In contrast to this,
492
similar studies conducted in cattle and rabbit suggested no effect of Stx on colonization in
493
intestine (49, 53). Thus the effect of stx2 gene and more specifically its subtype on
494
colonization in intestine needs more investigation.
495
CG strains induced more severe intestinal histopathological lesions when compared
496
with the BBG strains in the piglet and rabbit models, suggesting differences in their
497
ability to attach to and efface the intestinal mucosa. Recently, whole genome expression
498
microarray has been used to compare the differential gene expression between BBG and
499
CG strains (58). The important virulence factors, including the genes encoded on
500
chromosomal LEE, and several genes encoded on pO157 plasmid (the enterohemolysin
501
ehxA, toxB, and etp genes necessary for T2SS) showed increased expression in the CG
502
when compared with BBG strains. In contrast, the genes essential for survival in the
503
environment, such as acid resistance and stress response, were up regulated in the BBG
504
when compared with the CG strains (24, 55, 57, 58). This study supports our observations
505
suggesting that differential expression of LEE-encoded virulence genes in vivo have led
506
to the differences in the magnitude of intestinal bacterial load and histopathological
22
507
lesions in the intestines between the piglets and rabbits challenged with CG and BBG
508
strains. This initial damage to the intestinal epithelium may facilitate the absorption of
509
Stx into the blood stream where it may cause more severe systemic damage.
510
In conclusion, we have demonstrated that BBG and CG EHEC O157 differ in
511
virulence potential using two animal models, consistent with the hypothesis that not all
512
EHEC O157 strains are equally pathogenic. The amount of Stx and/or type of stx2 gene
513
present are correlated to the virulence differences of EHEC O157 genotypes and more
514
studies are needed in order to clarify the role of these bacterial factors in pathogenesis
515
and virulence of specific EHEC O157 genotypes.
516
517
ACKNOWLEDGEMENTS
518
This work was funded in part by NIAID NIH contract N01-AI-30055, R21AI073803, and
519
by the Agricultural Animal Health Program, Washington State University College of
520
Veterinary Medicine, Pullman, WA. The authors thank Rebecca A. King, Melissa W.
521
Mobley and Amanda Potter for their technical assistance with studies involving the rabbit
522
model.
523
524
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756
757
34
758
Table 1. Scoring system for clinical signs applied to neonatal piglets.
Diarrhea (consistency, frequency,
CNS signs
Vomiting
Loss of appetite
straining )
Abnormal
Vocalization
(0-3)
(0-5)
(0-1)
(0-2)
(0-1)
None (formed feces) (0)
None (0)
None (0)
None (0)
None (0)
Loose (not formed), no straining or
Lethargy (1)
Vomiting (1)
Slight (1)
Present (1)
blood (1)
Thick liquid, more than twice, mild
Splayleg, hind limb weakness,
straining and staining of hind limbs
thinner appearance (2)
Complete (2)
with feces (2)
Runny watery- with/without blood,
Shivering, tremors (3)
severe straining and staining of hind
limbs with feces (3)
Seizures, convulsions (4)
Grand mal seizures or lateral
recumbency or paralysis of legs (5)
759
35
760
Table 2. EHEC O157 strains used in this study and their origin.
EHEC Bank #
SBI Genotypea
CDC EDL 933
CG-3
Yearb
Source and Location of collectionc
Cohortd
Raw hamburger meat implicated in
hemorrhagic colitis outbreak
NA
E2325
CG-1
1995
Bovine fecal, WA USA
R1
E3046
CG-3
1995
Bovine fecal, WA USA
R3
E5252
CG-3
1998
Bovine fecal, OR, USA
R3
E5880
BBG-5
1999
Water, USA
R2
E6996
BBG-6
2000
Bovine fecal, WA, USA
R2
E12000 (Sakai)
CG-3
1996
Sakai City, Osaka Pref. Japan
NA
E12053
BBG-5
1993
Bovine fecal, USA
P3
E12056
BBG-5
1999
Water, USA
P1
E12057
BBG-6
1999
Bovine fecal USA
P2
E12058
BBG-6
2000
Bovine fecal, WA, USA
P1
E12059
BBG-5
2001
Bovine fecal, USA
P2
36
E12061
CG-1
2004
Human Clinical WADOH, WA USA
P2
E12062
CG-1
2004
Human Clinical WADOH, WA USA
P5
E12063
CG-3
2004
Human Clinical WADOH, WA USA
P2
E12064
CG-1
2005
Human Clinical WADOH, WA USA
P1
E12065
CG-3
2005
Human Clinical WADOH, WA USA
P3
E12066
CG-1
2006
Human Clinical WADOH, WA USA
P3
E12067
CG-3
2006
Human Clinical WADOH, WA USA
P1
E12068
BBG-5
1996
Bovine fecal, TX, USA
P5
E12149
BBG-6
1995
Bovine fecal, WA USA
P4
E12153
BBG-6
2001
Bovine fecal, WA, USA
P4
E12154
CG-1
2004
Human Clinical WADOH, WA USA
P5
E12155
CG-3
2006
Human Clinical WADOH, WA USA
P5
E12156
BBG-5
1994
Bovine fecal, WA, USA
P5
E12157
BBG-6
1995
Bovine fecal, OR, USA
P3
37
E12377
CG-3
2004
Human Clinical WADOH, WA USA
761
a
CG=Clinical genotypes, BBG=Bovine-biased genotypes
762
b
WADOH=Washington State Department Of Health
763
c
Year of the strain isolation or time deposited in WSU EHEC bank.
764
d
P=Litter cohorts of piglets, R= Experimental cohorts of Rabbits, NA= Not Assigned
P5
765
766
767
768
769
770
771
772
773
774
775
776
38
777
Table 3. Clinical scores, clinical scores for CNS disease, survival data, intestinal bacterial load and histopathological scores
778
for intestine in the piglets challenged with BBG strains, CG strains, positive (Sakai) and negative (sham-inoculated) control
779
piglets.
Disease Outcome
780
Groups of piglets (mean ± SEM)
BBG
CG
Negative
Positive
Clinical scores
1.5 ± 0.45 a
4.12 ± 0.66 b
0.98 ± 0.34 a
3.96 ± 0.26 ab
CNS disease Scores
0.69 ± 0.25 a
2.39 ± 0.46 b
0.07 ± 0.07 a
2.35 ± 0.28 b
Survival time (days)
6.20 ± 0.55 a
3.70 ± 0.06 b
7.00 ± 0.00 a
4.25 ± 0.63 ab
Intestinal bacterial load (cfu)
4.30 ± 0.18 a
5.09 ± 0.19 b
1.13 ± 0.47 c
5.85 ± 0.17 b
Histopathological scores of intestine
1.08 ± 0.23
2.01 ± 0.30
1.00 ± 0.52
2.88 ± 0.58
Groups not sharing superscript letters differed significantly (P≤0.05).
39
781
Table 4. Intestinal and renal lesions in rabbits infected with various Escherichia coli
782
O157:H7 strains*
783
784
785
O157:H7
strain
Intestinal
vasculopathy
Capillary
thickening
Luminal
constriction
Erythrocyte
fragmentation
Intimal
swelling
Perivas
edema
EDL933
E5252
P<0.002
P<0.003
NS
P<0.03
NS
P<0.008
E3046
P<0.01
NS
NS
P<0.008
(P<0.006)
NS
NS
P<0.003
(P<0.0003)
P<0.03
(P<0.004)
NS
NS
NS
NS
P<0.02
(P<0.00
P<0.04
(P<0.00
NS
NS
NS
NS
E2325
P<0.007
NS
NS
NS
E6996
P<0.04
NS
NS
NS
E5880
NS
NS
NS
P<0.01
*, P values are relative to sham-inoculated controls; NS, not significant; P values in
parenthesis are relative to EDL933-infected rabbits.
786
787
788
789
790
791
792
793
794
795
796
797
798
Figure 1. Clinical scores of piglets challenged with EHEC strains belonging to different
799
genotypes. Groups not sharing specific letters differed significantly (P=0.023).
40
6
5
4
3
2
1
0
CG-1
800
801
CG-3
BBG-5
BBG-6
Genotype
802
803
804
805
806
41
807
Figure 2. Kaplan-Meier survival curves for piglet groups challenged with different
808
EHEC O157 genogroups.
1.0
0.8
0.6
0.4
0.2
BBG
CG
Negative (Sham-inoculated)
Positive (Sakai)
0.0
0
809
810
1
2
3
4
5
6
7
Time (Days)
811
812
813
814
42
815
Figure 3. Piglet challenged with positive control Sakai strain. (A) Section of the brain
816
stained with LFB stain showing affected blood vessel (arrows, magnified in fig. 3B and
817
3C) with vacuolation of adjacent tissues (arrow head) caused by focal myelin
818
degeneration. (B) Affected blood vessel in brain showing microhemorrhage (long arrow),
819
pyknosis (short arrow) and hyperplasia (arrow head). (C) Affected blood vessel in brain
820
showing perivascular edema. Bar=100µm.
821
822
823
824
43
825
Figure 4. Serosal hemorrhage in the area of the distal cecum adjacent to the junction
826
with the proximal colon in a Dutch Belted rabbit infected with Escherichia coli O157:H7
827
strain EDL933.
828
829
830
831
832
833
834
835
836
837
838
839
840
841
842
44
843
Figure 5. A, Sham-inoculated normal rabbit cecum. B, O157:H7 strain E5252-infected
844
rabbit cecum with submucosal edema and necrotizing vasculitis (arrow). C, Higher
845
magnification of submucosal vascular lesion in “B” showing heterophilic vasculitis and
846
perivasculitis with fibrinoid vascular degeneration/necrosis and intimal proliferation. D,
847
normal rabbit glomerulus. E, Glomerulus of a rabbit infected with O157:H7 strain E5252
848
demonstrating mild capillary thickening (arrows). F, Glomerulus of a rabbit infected with
849
O157:H7 strain E5252 showing global edematous swelling, luminal constriction,
850
decreased numbers of erythrocytes (“bloodless glomerulus”), and fragmentation of
851
erythrocytes (arrow).
852
853
854
855
856
857
45
858
Figure 6. Stx ELISA OD450 scores by E coli O157:H7 genotypes following uninduced,
859
nalidixic acid induced, or carbadox induced growth in broth enrichment culture. All
860
strains are nalidixic acid resistant. .
4
Un-induced
Nalidixic acid induction
Carbadox induction
3
2
1
0
CG-1
861
862
CG-3
BBG-5
BBG-6
Genotype
863
864
865
866
46
867
868
869
870
871
47