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African Journal of Pig Farming ISSN 2375-0731 Vol. 5 (11), pp. 001-005, November, 2017. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 

 

Full Length Research Paper 

 

Escherichia coli O157:H7 EDL933 has a strong 

virulence to Bama miniature pigs by injection and 

fails to colonize to their gastrointestinal tracts 
 

Honglei Ding1,2,3, Rui Zhang1, Kaiyun Liu1, Linping Huang1, Maochun Tian4, Mingming 
Jiang1, Quanming Zou1 and Xuhu Mao1* 

 
1
Department of Clinical Microbiology and Immunology, Faculty of Medical Laboratory Science, Third Military Medical 

University and National Engineering Technological Research Center of Immunological Biologicals, Chongqing, China. 
2
Institute of Sericulture and System Biology, Southwest University, Chongqing, China. 

3
Chongqing Productivity Council, Chongqing, China. 

4
College of Animal Science and Technology, Southwest University, Chongqing, China. 

 
Accepted 21 July, 2017 

 
Detection of Shiga toxin-producing Escherichia coli O157:H7 from commercially grown pigs has been reported. 
Furthermore, the E. coli O157:H7 colonized model of pig has been established and E. coli O157:H7 could be 
transmitted from infected donor pigs to naïve pigs directly and indirectly. In the present study, we want to know 
whether any E. coli O157:H7 strain can colonize to the alimentary tract of pig and the virulence of E. coli 
O157:H7 to pig by injection. Bama miniature pig was infected with E. coli O157:H7 EDL933 strain orally, but the 
organism could not be recovered from the feces and did not cause any tissue damage. Nevertheless, this 
pathogen introduced serious clinical symptoms and pathological injuries by injection, especially the nervous 
system and the injected pig exhibited severe neurological symptoms, including synclonus tremens, ataxia, 
head-pressing and recumbency, etc. The pig did not excrete urine and feces and the abdomen became 
tympanous. These data suggested that only certain E. coli O157:H7 strains could colonize to the GIs of pigs 
involved mechanisms that related to various factors. However, the organism has strong virulence to pig by 
injection mode and it is a risky pathogen to human health. 
 
Key words: Escherichia coli O157:H7, Bama miniature pig, colonization, inoculation, pathological injury. 
 
INTRODUCTION 
 
Infection of Shiga toxin -producing Escherichia coli 
(STEC) O157:H7 can lead to a spectrum of illnesses in 
human, including diarrhea, hemorrhagic colitis (HC) and 
hemolytic uremic syndrome (HUS), which demonstrates 
as acute renal failure and may lead to death (Besser et 
al., 1993; Bruce et al., 2003; Rivas et al., 2006).  

Most cases are thought to occur as a result of the 
ingestion of ground beef (Bell et al., 1994), unpasteurized 

milk (Solomakos et al., 2009) and vegetables (Besser et 

al., 1993), which are thought to have been contaminated  
 
 
 
*Corresponding author. E-mail: maoxuhu@163.com, 
mxh95xy@mail.tmmu.com.cn. Tel: 86-23-68752315. Fax: 86-

23-68752315. 

 
 
 

 
with feces from infected cattle. Outbreaks and sporadic 
cases have also been linked to water, animal-to-person 
and person-to-person transmission (Swerdlow et al., 
1992; Belongia et al., 1993; Shukla et al., 1995).  

Cattle are considered to be the major reservoir of STEC 
and the prevalence of E. coli O157:H7 in cattle is range 

(Baker et al., 2007; Wang et al., 2008; Williams et al., 
2008). E. coli O157:H7 has also been isolated from other 
ruminants, such as deer (García-Sánchez et al., 2007; 
Sánchez et al., 2009) and sheep (Kudva et al., 1996). E. 
coli O157:H7 has occasionally been isolated from non-
ruminant animals, including poultry (Baschkier et al., 
2009; Heuvelink et al., 1999), pigeons (Cízek et al., 2000; 
Kobayashi et al., 2002), wild birds (Wallace et al., 1997; 
Kobayashi et al., 2002) and raccoons (Hancock et al., 
1998), but the bulk of the data suggests that the, 



 
 
 

 

prevalence of STEC is greater in ruminants than in other 
animals.  

Recent studies have demonstrated that conventional 
pigs were permissive host for E. coli O157:H7 (Booher et 
al., 2002; Cornick and Helgerson, 2004) and have esta-
blished that E. coli O157:H7 could be shed by 3- month-
old pigs for 2 months. Furthermore, these animals did not 
become clinically ill, and the duration of shedding in the 
feces was similar to that of ruminants experimentally 
infected with the same E. coli O157:H7 isolate (Booher et 
al., 2002) and could transmit this pathogen to their 
offspring. Meanwhile, some researches indicated that E. 
coli O157:H7 had been isolated from healthy pig in many 
countries (Heuvelink et al., 1999; Wang et al., 2008; 
Oporto et al., 2008).  

Nevertheless, the prevalence of the organism in these 
studies was generally low, except for the result from Chile 
that the prevalence of E. coli O157:H7 was higher in pigs 
than in cattle, which suggested that pig might be an 
important source of this organism in some countries 
(Borie et al., 1997). Also, only one family outbreak has 
been specifically traced back to pork salami and the E. 
coli O157 isolated from the couple and the salami carried 
Shiga toxin 1 (stx1), Shiga toxin 2 (stx2) and E. coli 
attaching and effacing (eae) genes and shared the same 
PFGE (pulsed- field gel electrophoresis) pattern 
(Conedera et al., 2007) . To date, there is no explanation 
for the reason why the prevalence of E. coli O157:H7 in 
pig is generally lower than in ruminants. Also, no 
research about the consequence of pig infected this 
organism in its organs or abdomen, though these animals 
did not become clinically ill infected this pathogen orally.  

We hypothesized that the gastrointestinal tract (GI) of 
pig did not facilitate the colonization of most E. coli 
O157:H7 strains and caused the low prevalence of E. coli  
O157:H7 in pig. In the study, E. coli O157:H7 EDL 933 

was selected, which obtained from a patient whose 
symptom, characterized by severe cramped abdominal 
pain, initially watery diarrhea and grossly bloody diarrhea 
(Riley et al., 1983) and determined whether it was a 
suitable strain for the colonization to the GI of pig. Also, 
the virulence of E. coli O157:H7 to pig was detected by 

injection. 
 

 
MATERIALS AND METHODS 
 
E. coli O157:H7 strain 
 
E. coli O157:H7 strain EDL933 (stx1, stx2, eaeA, ehxA, Tccp and 
espA positive) was originally isolated from patient with diarrhea 
(Riley et al., 1983) and was kindly provided by Prof. Huaiqi Jing 
(Chinese Center for Disease Control and Prevention). E. coli 
O157:H7 CYB42 was isolated from diary cattle in Chongqing (Wang 
et al., 2008). These bacterial inocula were grown as previously 
described with minor modification (Booher et al., 2002). Briefly, a 
single colony from strain was picked and cultured overnight in Luria-
Bertani (LB) medium at 37°C with shaking (180 rpm) and the 
bacterial number was confirmed by direct plate counts. The inocu-
lum was washed once with 0.1 M phosphate buffered saline (PBS) 

  
  

 
 

 
(pH 7.2) and adjusted to the appropriate concentration. 

 

Animals and preparation 
 
Bama miniature pigs were obtained from a commercial source at 1 
month of age and housed in pens with cement floors at 2 pigs per 
pen under biohazard level 2 facilities. The pigs were acclimated to 
an antibioticfree feed (creep feed, Chongqing Zhengda Company, 
Chongqing) and water ad libitum for 2 weeks prior to inoculation. 
Fecal samples were collected from each animal once prior to 
inoculation and injection and screened with sorbitol-MacConkey 

agar supplemented with cefixime (2.5 mg
.
l 

-1
) and potassium 

tellurite (0.05 mgl
-1

) (CT- SMAC) and polymerase chain reaction 
(PCR) to ensure that the pigs were not naturally colonized by E. coli 
O157:H7. 

 

Inoculation of E. coli O157:H7 
 
18 pigs were used in inoculation and were separated into 3 groups, 
of which 16 were inoculated with the EDL933 and 2 were negative 
controls. After a 2 week acclimation period, groups 1 and 2 of each 

8 pigs were inoculated with 1.0 × 10
8
 and 1.0 × 10

12
 clonal forma-

tion unit (CFU) of E. coli O157:H7 EDL933 by adding the organism 
to a small amount of food placed in individual pans, respectively. 
On the 17th day after fed EDL933 bacteria, groups 1 and 2 of each 

4 pigs, selected randomly, were inoculated with 1.0 × 10
8
 and 1.0 × 

10
12

 CFU of E. coli O157:H7 CYB42. Group 3 of 2 pigs were 
inoculated with 10 ml 0.1 M PBS (pH 7.2). Pigs were observed until 
the inoculum was consumed. 

 

Injection of E. coli O157:H7 lysate 
 
18 pigs were used in injection and were also separated into 3 groups. 
After a 2 week acclimation period, group 1 of 8 pigs were injected with 

1.0 × 10
8
 CFU of E. coli O157:H7 strain EDL933 lysate intramuscularly; 

group 2 of 8 pigs were injected with 1.0 × 10
8
 CFU E. coli O157:H7 

EDL933 lysate intraperitoneally; and group 3 of 2 pigs were injected 
with 10 ml 0.1 M PBS (pH 7.2) intramuscularly. 

 

Fecal sampling 
 
Individual fecal samples from inoculated pigs were collected on 
days 2, 3, 4 and at 2 weeks post inoculation (pi) (days 14, 15, 16) of 
EDL933 and CYB42, respectively, and from injected pigs were 
collected on days 2, 3, 4 (Booher et al., 2002). Fecal samples were 
cultured as previously described (Cornick and Helgerson, 2004). 
Briefly, 5 g samples were added to 20ml of PBS (pH 7.2) and mixed 
in a Stomacher blender, and then serial 10 fold dilutions were made 
to use PBS (pH 7.2).  

Samples were directly inoculated in triplicate into selective media 
CT-SMAC. Enrichment cultures (10 g of feces in 100 ml LB plus 
0.02% bile salts) were incubated overnight at 37°C, concentrated 
using immunomagnetic beads (Dynabeads; Dynal, Oslo, Norway), 
and plated into the selective medium described above. The 
sensitivity of the direct plating method was 50 CFU/g. Colonies 
recovered on selective medium were confirmed as E. coli O157:H7 
by using a commercial latex agglutination kit specific for the O157 
lipopolysaccharide and PCR for rfbE (Wang et al., 2008). 

 

Necropsy 
 
The animals monitored closely to ensure their welfare after they 

were injected. Both control and E. coli EDL933 injected animals 



 
 
 

 

were euthanized and necropsied at the 4
th

 day when they behave 

severe clinical signs. Meanwhile, after 2 weeks pi, control, E. coli 
EDL933 (8 pigs) and E. coli CYB42 (8 pigs) inoculated animals 
orally were euthanized and necropsied. Pigs were sedated with an 
injectable anesthetic (tiletamine HCl and zolazepaam HCl) and then 
euthanized with an intravenous overdose of sodium barbiturate 
(390 mg pentobarbital sodium and 50 mg phenytoin sodium/5 kg of 
body weight) (Booher et al., 2002).  

The following types of tissue (approximately 5 cm length or 5 cm 
square) were collected from pigs: stomach, jejunum, ileum, distal 
colon, cecum, rectum, epencephalon, cerebrum, kidney, liver, and 
spleen. The sections of tissues were collected in neutral buffered 
formalin for histopathology, processed, and stained with 
hematoxylin and eosin (H and E). 5 g of rectal contents were also 
collected and cultured by using direct plating and enrichment broth 
as described above. 

 

Western blotting 
 
Serum samples were collected from both control and E. coli 
inoculated pigs prior to inoculation and at necropsy and detected 
stx2, intimin and espA neutralizing antibodies. The stx2, intimin and 
espA recombinant purified proteins were prepared in our lab (Gu et 
al., 2009; Ma et al., 2008). SDS-PAGE and western blotting were 
performed as described previously (Cendron et al., 2009). For 
visualization of proteins after SDS-PAGE, gels were stained with 
Coomassie brilliant blue R250.  

For the development of immunoblots, PVDF filters were blocked 

with blocking buffer (Beyotime) and incubated with the respective 
antisera at a dilution of 1:1000. The membrane was washed 6 times 

with TBS containing 0.1% Tween-20 (TTBS) (pH 7.5). Horseradish 
peroxidaseconjugated anti-rabbit IgG was used at a dilution of 1:10 

000 to visualize bound antibody. 
 

 

RESULTS 

 

Clinical response and bacterial culture of inoculated 

pigs 
 
None of the animals developed signs of intestinal or 
systemic disease following inoculation with the E. coli  
inoculum from the 2 groups that were inoculated the strain 
orally. E. coli O157:H7 was not recovered from any of the 16 
pigs during the initial period or at 2 weeks after inoculation with  
1.0 × 10

8
 and 1.0 × 10

12
 CFU. Fecal samples from the rectal-

anal junction were also collected from these 16 pigs and no 
target organism was discovered.  

Then, groups 1 and 2 of each 4 pigs were inoculated 

with 1.0 × 10
8
 and 1.0 × 10

12
 CFU of E. coli O157:H7 

CYB42. However, E. coli O157:H7 CYB42 was still not 
recovered from the 8 inoculated pigs. No E. coli O157:H7 
was investigated from any of the two control animals at 
any time during the experiment, and no symptom 
displayed. 
 

 

Clinical observation and bacterial culture of injected 

pigs 
 
Piglets challenged intramuscularly and intraperitoneally 

with EDL933 typically developed neurological signs within 

 
 
 
 

 

36 and 48 h, respectively, including anorexia, depression  
and paralysis of the hind limbs like goggy sitting. However, 
the feces are dry. 48 to 72 h following injection with E. coli  
O157:H7 strain, the piglets lay on one side, and went on 
to manifest severe neurological symptoms of synclonus 
tremens, ataxia, head-pressing and recumbency. Simul-
taneously, the mouth was slightly open and jerked 
violently and spasmodically and the 2 fore limbs stroked 
like swimming. The pig did not excrete urine and feces 
and the abdomen became tympanous.  

When the anocelia was opened, a little of serous fluid 
leaked from the abdominal subcutaneous tissue, and the 
livers and spleen were adhered to the peritoneum. The 
intestinal wall was thinner than the normal one. There 
was too much urine in the bladder, which caused the 
tympanous abdomen. The blood became thick and 
reddish black with slow bloodstream.  
No stx2, intimin and espA neutralizing antibodies were 
detected in both E. coli O157:H7-infected and E. coli 

O157:H7-injected pigs. 

 

Histological studies on E. coli O157:H7-infected and 

E. coli O157:H7-injected pigs 
 
We also studied the effect of E. coli O157:H7 injection on 
their main target organs, including stomach, intestine, 
epencephalon, cerebrum, kidney, liver and spleen by 
examining H and E stained-sections. The main 
pathological damage was observed at cerebrum (Figure 
1), epencephalon (Figure 2) and large intestine (Figure  
3). The nerve cells of cerebrum became spindle-shaped 
and hydropic, accompanying coagulation necrosis and 
the nucleus was pycnotic. In addition, blood capillary was 
atresic, and perivascular space was broadened.  

Examination at epencephalon revealed the obvious 
reduction of cells in tunicae granulosa and neuropile 
porous. The members of the research group could also 
detect karyopycnosis of purkinje cell. Histological exami-
nation of ceca, recta of E. coli O157:H7 injected pigs 
showed multi- focal areas of villous or surface epithelium 
degeneration, necrosis, or shedding, submucous 
membranous hydropsia, and extensive inflammatory cell 
infiltration, mainly the lymphocyte in proper coat. Sur-
prisingly, the small intestine and kidney (Figure 4) only 
showed slightly pathological change with mild cell trauma. 
For instance, some glomeruli of kidney increased in 
volume, and the capsular space became narrow.  

Proximal convoluted tubule cells exhibited denaturation 

and necrosis. Meanwhile, the blood capillary was 
enlarged. Conversely, E. coli O157:H7-infected group of 

pigs did show any pathology in their tissues. There were 
no abnormalities in the control animals as well. 

 

DISCUSSION 
 
During the experiment, it was found that EDL933 could 

not infect Bama miniature pigs orally, though the 



 
 
 

 

infectious dose is high. In order to further confirm this 
outcome, the researchers chose E. coli O157:H7 CYB42 
that was isolated from diary cattle in Chongqing 
toreinoculate the EDL933-feeded pigs. But, the result was 
negative as previously. It demonstrated, to some extent, 
that some pig individuals were resistant to colonization 
and/or some E. coli O157:H7 strains could not colonize to 
the intestinal epithelium of pig. Some researches 
indicated that many bacterial factors contributed to the 
colonization.  

Some research data demonstrated that the flagellum of 
E. coli O157:H7, but not intimin, was important for 
persistence in poultry (Best et al., 2005), whereas intimin 
(Woodward et al., 2003) but not the flagellum was 
important in conventionally weaned lambs. This suggests 
that the function of both surface arrayed structures may 
be host dependent and may be linked to the availability of 
specific host-cell receptors. Best et al suggested that the 
flagellum and intimin of a stx -negative E. coli O157:H7 
isolate had little or no role to play in colonization of 14 
week-old conventionally reared pigs (Best et al., 2006).  

However, other reported E. coli O157:H7 virulence 
factors, such as long polar fimbriae, did contribute to the 
persistence in pig animal infection model (Jordanet et al., 
2004), and might have contributed to the persistent 
infection of pigs noted for the intimin and flagella deficient 
mutants reported. The strain, 86 - 24, which was isolated 
from an outbreak of human disease and caused attaching 
and effacing lesion, was used in most animal experiments 
and considered as a well established E. coli O157:H7 
infected pathogen. The EDL933 strain, but not 86 - 24, 
may be absent from the essential factors, which were 
necessary for the long-term colonization of the pigs' 
intestinal tracts. This could partially explain why EDL933 
could not adhere to Bama miniature pigs' GI. The genetic 
difference of the 2 strains for colonization in animals 
needs further comprehensive researches.  

In this experiment, we also attempted to establish a 
Bama miniature pig model infected steadily with E. coli 
O157:H7, for the small weight of the Bama miniature pig 
of which the figure is thinner than susscrota domestica's. 
Nevertheless, the EDL933 strain could not colonize to the 
GIs of Bama miniature pigs. In the next plan, the research 
group will try to inoculate and acclimate EDL933 strain 
into Bama miniature pig through certain methods, such as 
serial passages in vivo, for procuring an adaptive 
colonization of E. coli O157:H7 strain.  

To identify whether E. coli O157:H7 was pathogenic to 
pigs, nonproliferative EDL933 lysate were injected to 
Bama miniature pigs intramuscularly and intraperi-
toneally. Interestingly, the infected pigs exhibited identical 
clinical symptom, especial the nervous syndrome. 
However, the injected pigs did not manifest diarrhea, 
which was consistent to the histological findings. Serious 
intestinal microvillus damage may be an indispensable 
process caused through intestinal infection of E. coli 
O157:H7. Therefore, E. coli O157:H7 will be pathogenic  
when it enters the organa parenchymatosums or abdomens 

  
  

 
 

 

of pigs. 
 

 

Conclusion 
 
Collectively, only certain E. coli O157:H7 strains could 

colonize to the GIs of pigs. It explained the low 
prevalence of E. coli O157:H7 in pigs partially. However, 

this organism has strong virulence to pig by injection 

mode, and it is a risky pathogen to human health. 

 

ACKNOWLEDGEMENTS 
 
This work was supported by the 863 high-tech project of 

China (2006AA02Z443) and the national science 

foundation of China (30970116). 

 
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