




































In ternationa l
Scholars
Journa ls

 

African Journal of Pig Farming ISSN 2375-0731 Vol. 3 (7), pp. 001-004, July, 2015. Available online at 
www.internationalscholarsjournals.org © International Scholars Journals 

 

Author(s) retain the copyright of this article. 

 
 

Full Length Research Paper 

 

Prevalence of (high-pathogenicity island) HPI-harboring 
Escherichia coli in diarrheic and healthy piglets 

 
DaRong Cheng1*, XiaoLang Chen1, ShanYuan Zhu2, WenWei Ding1, ZhiXia Mu1 and Jun Zhou2

 
 

1
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China. 

2
Jiangsu Animal Husbandry and Veterinary College, Taizhou 225300, China. 

 
Accepted 22 October, 2014 

 
One hundred and sixty four rectal swab samples were collected from 110 live diarrheic piglets and 54 healthy piglets 
during an investigation on the prevalence of HPI-harboring Escherichia coli (HPI 

+
 E. coli) infection associated with 

porcine diarrhea. The data of polymerase chain reaction (PCR) revealed the presence of HPI 
+
 E. coli in 58.18% of swabs 

taken from diarrheic piglets, while 44.44% of samples taken from healthy pigs were so positive. Further PCR examination 
of 600 bacterial isolates from diarrheic samples revealed that 25 isolates were HPI 

+
 E. coli while 4 (16%) were F4

+
, 2 (8%) 

were F4
+
 and F6

+
, 2 (8%) were F4

+
 and F6

+
, and one (4%) was F6

+
, LTa

+
 and STb

+
. Among the 480 bacterial isolates from 

non-diarrheic samples, 20 isolates were HPI 
+
 E. coli, and only one (5%) was LTa

+
 and STb

+
, while absence of isolates 

belonging to the F4
+
 and F6

+
 groups was noted. O138 was the vast prevalent serotype among the HPI 

+
E. coli isolates. It 

is suggested that HPI 
+
 E. coli maybe an opportunistic pathogen in swine. 

 

Key words: Escherichia coli, HPI, prevalence, piglet. 

 
INTRODUCTION 

 
Pathogenicity islands (PAIs) are a distinct class of 
genomic islands (GEIs), which are acquired by horizontal 
gene transfer. PAIs harbor virulence genes and some 
antibiotic resistance genes (Oelschlaeger et al., 2004). 
The high-pathogenicity island (HPI) was previously 
described in Yersinia pestis, Yersinia pseudotuberculosis 
and Yersinia enterocolitica (Bearden et al., 1998; 
Buchrieser et al., 1998a).  

In addition, HPI have been horizontally transferred to 
other bacterial species, such as Shigella, citrobacter, 
Klebsiella and Escherichia (Bach et al., 2000; Karch et 
al., 1999; Schubert et al., 1998). It was also reported that 

some HPI
+
 E. coli isolates from patients or animals with 

diarrhea were closely correlated with clinical symptoms 
(Xu et al., 2000; Cheng et al., 2006).  

But the role of HPI 
+
 E. coli isolates play in the piglet 

diarrhea need to be deeply investigated and researched 

in detail. In this study, we detected HPI 
+
 E. coli infection 

in diarrheic and healthy piglets, and investigate the pre-

valence of the other virulence factors in the HPI 
+
 E. coli 

isolates.  
 
 
 
*Corresponding author. E-mail: jsyzcdr@yahoo.com.cn. 

 
 
 
 
 

 
MATERIALS AND METHODS 
 
Collection of samples 
 
From March - June, 2009, a total of 164 rectal swab samples were 
collected from piglets with a mean age of 6.5±3.4 days, including 
110 live diarrheic piglets from 28 swine farms and 54 healthy 
piglets from 18 swine farms in the Jiangsu Province, China. This 
brings each test sample to a final volume of 500 μl with LB broth 
(10 g tryptone, 10 g NaCl, 5 g yeast extract, H2O to 1 L, pH 7.4) 
and store at -70°C. 

 
Bacterial pre-culturing and extraction of DNA templates 
 
100 μl dilution of each rectal swab sample was transferred into 
separate tubes containing 2 ml of LB broth and the liquid cultures 
were grown with vigorous agitation at 37°C for 6 h. 0.5 ml of the 
liquid cultures was transferred to labeled micro centrifuge tubes, 
and centrifuged at 10,000 rpm for 5 min. The supernatants were 
discarded and each pellet was resuspended in 200 μl of distilled 
water. After boiling for 10 min, the suspensions were chilled on ice 
for 5 min, centrifuged at 10,000 rpm at 4°C for an additional 5 min, 
and the supernatants used directly or stored at 4°C before PCR 
detection. 

 
PCR detection of HPI-harboring E. coli 
 
To detect HPI-harboring E. coli in the liquid cultures of rectal swab 



       
 

 Table 1. Summary of the O serotype and virulence factors of the 25 HPI 
+
 E.coli isolates from diarrheic piglets. 

 

         
 

  Virulence factor      
Numbers of 

 

   
LTa STa STb F4 F6  

   isolates  

  

O serotype 
     

 

        
 

  O138 - - - - - 12 
 

  O138 - - - - + 2 
 

  O138 - - - + + 1 
 

  O138 + - + - + 1 
 

  O65 - - - - - 3 
 

  O139 - - - - + 2 
 

  O9 - - - + - 2 
 

  O55 - - - + + 1 
 

  O141 - - - - - 1 
 

  Total 1 0 1 4 7 25 
  

 
 

 
samples, PCR assays were performed in micro centrifuge tubes for 
the Applied Biosystems (2720 Thermal Cycler America). The  
primers (F: 5’-AAGGATTCGCTGTTACCGGAC-3’, R: 5’-
TCGTCGGGCAGCGTTTCTTCT-3’) were used for PCR amplifi-cation 

to detect HPI 
+
 E. coli (Schubert et al., 1998) and synthesized by 

Sangon Biological Engineering Technology and Service Co. Ltd. 
(Shanghai, PR China). All the PCR reagents were purchased from 
TaKaRa Biotechnology (Dalian) Co., Ltd. The PCR mixture contained 5 

μl of 10× PCR buffer (Mg
2+

 plus), 5 IU of Taq poly-merase, 4 μl of dNTP 

mixture (each 2.5 mmol/l), 1 μl of HPI primers set (each 50 mmol/l), 2 μl 
of DNA template, and deionized water to a final volume of 50 μl. After 
denaturation at 94°C for 3 min, 30 cycles of the PCR was performed 
using the following program: denature-tion at 94°C for 30 s, annealing at 
62°C for 30 s and elongation at 72°C for 60 s (10 min for the final cycle). 
The expected size of PCR products should be 287 bp following 
separation in 1% agarose gel electrophoresis along with DL2000 DNA 
markers and visualization on a UV transilluminator after staining with 
ethidium bromide. 
 
 
Virulence genes determination and O serotyping of HPI-
harboring E. coli 
 
Following rapid PCR detection, 30 HPI-positive diarrheic samples 
and 24 HPI-positive healthy samples were submitted to separative 
cultivation of bacteria by streaking on Mackonkey agar plates. After 
incubation at 37°C overnight, twenty colonies were picked from 
each sample and cultured on LB agar plates at 37°C for 24 h. All 
the bacterial isolates were submitted to PCR detection (Cheng et 
al., 2005b, 2006) for the genes of HPI, enterotoxins (STa, STb and 
LTa) and fimbriae (F18, F4, F5, F6 and F41). In addition, all the 
HPI-harboring E. coli were serotyped by using uni-factor serum of O 
antigen of E. coli. 
 

 

RESULTS 

 

Among the 110 rectal swab samples from live diarrheic 
piglets, 64 (58.18%) samples (from 22 farms) were 
infected with HPI-harboring E. coli, while 24 (44.44%) 
samples (from 9 farms) were HPI-positive in the 54 
healthy samples.  

Six hundred bacterial isolates were picked out from the 
diarrheic samples, and the data for PCR examination 

 
 
 

 
determined that only 25 isolates (from 22 samples) were 

HPI 
+
 E. coli, thereinto, 4 (16%) were F4

+
, 2 (8%) were 

F4
+
 and F6

+
, and 1 (4%) was F6

+
, LTa

+
 and STb

+
 (Table 

1). In addition, the 25 HPI 
+
 E. coli isolates were O 

serotyped and O138 was the most prevalent serotype 
accounting for 64% (16/25), followed by O65 (12%), O139 
(8%), O9 (8%), O55 (4%) and O141 (4%) (Table 1).  

Among the 480 bacteria isolates that were picked out 
from the non-diarrhoeic samples, just 20 isolates (from 

18 samples) were HPI 
+
 E. coli, thereinto, only one (5%) 

was LTa
+
 and STb

+
, while no single F4

+
 and F6

+
 isolate 

was detected (Table 2). Furthermore, the 20 HPI 
+
 E. coli 

isolates were O serotyped and O138 also was the most 
prevalent serotype accounting for 60% (12/20), followed 
by O65 (25%), O21 (10%), O9 (5%), and O74 (5%) 
(Table 2). 
 

 

DISCUSSIONS 

 

HPI was first discovered in pathogenic Yersinia strains 
and has recently been found to be widespread in other 
enterobacteria (Buchrieser et al., 1998b; Hacker et al., 
2000; Carniel et al., 1992; Fetherston et al., 1994; Perry 
et al., 1990; Schubert et al., 1998). HPI carries the gene 
fyuA, which is specific for the pesticin receptor (FyuA) 
and the irp (iron repressible protein, such as HMWP1 and 
HMWP2) loci encoding the siderophore yersiniabactin 
(Carniel et al., 1992; Guilvout et al., 1993; Lucier et al., 
1996). The HPI element is associated with asparagine-
specific tRNA loci and carries an integrase gene int, often 
associated with a phage genome (Buchrieser et al., 
1998; Rakin et al., 2001). But whether HPI could 
contribute to the virulence of E. coli isolates responsible 
for piglet diarrhea remains deeply a research question. 
 

In this research, the data revealed the presence of HPI 
+
 E. coli in 58.18% of swabs taken from diarrheic piglets, 

while 44.44% of samples taken from healthy pigs were 



 
 
 

 

Table 2. Summary of the O serotype and virulence factors of the 20 HPI 
+
 E.coli isolates from non-diarrheic piglets.  

 
 Virulence factor      

Numbers of 
 

  
LTa STa STb F4 F6  

  isolates  

 

O serotype 
     

 

       
 

 O138 - - - - - 10 
 

 O138 + - + - - 1 
 

 O65 - - - - - 5 
 

 O21 - - - - - 2 
 

 O9 - - - - - 1 
 

 O74 - - - - - 1 
 

 Total 1 0 1 0 0 20 
 

 

 

highly positive. Therefore, we are suspicious of whether 
HPI could contribute to the virulence of E. coli isolates 
that causes piglet diarrhea. But after the analysis on the 
other hand, it was found that 78.57% diarrheic farms 

were infected with HPI 
+
 E. coli, while 50.00% healthy 

farms were confirmed to be HPI-positive. Furthermore, 

some HPI 
+
 E. coli isolates from diarrheic samples more 

frequently contained other virulence-associated genes, 
such as toxin(s) and fimbria(e), while only one isolate 

from healthy samples was LTa
+
 and STb

+
, although the 

preva-lent O serotype all was O138 in diarrheic and 
healthy isolates. According to the previous investigation 
on the effects of different virulent factors of Escherichia 
coli on pathogenesis (Cheng et al., 2005a), it suggested 

that the HPI 
+
 E. coli most likely to be opportunistic 

pathogen. This attractive hypothesis has to be verified by 
comparing the virulence of the parental strain and that of 
the isogenic mutants in a suitable infection mode.  

It is well known that most E. coli are the normal inhabitants 

of intestinal tracts of animals (Levine, 1987; Martins et al., 

2000), and it is very difficult to obtain the pathogenic E. coli 

in the rectal swab samples from live diarrheic piglets by 

bacteria isolation and identification. As a matter of fact, the 

clinical diagnosis of E. coli infection in piglets, especially in 

the rectal swab samples from live diarrheic animals, usually 

just need confirming whether the pathogenic E. coli 

was/were existed in the sample(s), and not always need 

bacterial isolation and identification anymore, while it is so 

difficult to pick out the pathogenic isolate(s). This is perfectly 

supported by the data of this research, such as 58.18% 

diarrheic rectal swab samples could be confirmed to be HPI-

positive by the rapid detection method, and only 22 HPI 
+
 E. 

coli were obtained in 600 bacteria isolates by bacteria 

isolation and identification; while most isolates were the 

important members of the normal microbiologic flora of 

piglets. The experience of this research could provide a 

constructive idea and use for the diagnosis of other 

pathogen infection. 
 
 
ACKNOWLEDGEMENT 

 

This work was supported by National Natural Science 

 

 

Foundation of China (Grant No. 30800821) and Jiangsu 
Province Key Laboratory of High Technology 
Researching Program (Grant No. BM2009701). We 
would like to give our thanks to all the staff of the 
veterinary microbiology laboratory of Yangzhou 
University for their help with some experiments. 

 
REFERENCES 
 
Bach S, de Almeida A, Carniel E (2000). The Yersinia high-

pathogenicity island is present in different members of the family 
Enterobacteriaceae. FEMS Bicrobiol. Lett., 183: 289-294.  

Bearden SW, Fetherston JD, Perry RD (1997). Genetic organization of 
the yersiniabactin biosynthetic region and construction of avirulent 
mutants in Yersinia pestis. Infection Immunity, 65: 1659-1668.  

Buchrieser C, Prentice M, Carniel E (1998a). The 102-kilobase unstable 
region of Yersinia pestis comprises a high-pathogenicity island linked 
to a pigmentation segment which undergoes internal rearrangement. 
J. Bacteriol., 180: 2321-2329.  

Buchrieser C, Brosch R, Bach S, Guiyoule A, Carniel E (1998b). The 
high pathogenicity island of Yersinia pseudotuberculosis can be 
inserted into any of the three chromosomal asn tRNA genes. 
Molecular Microbiol., 30: 965-978.  

Carniel E, Guiyoule A, Guilvout I, Mercereau-Puijalon O (1992). 
Molecular cloning, iron-regulation and mutagenesis of the irp2 gene 
encoding HMWP2, a protein specific for the highly pathogenic 
Yersinia. Molecular Microbiol., 6: 379-388.  

Cheng D, Huang W, Zhang Y, Zhang K, Xu J (2005a). The detemintion 
of virulence of seven strains of different virulent of Escherichia coli. 
Progress in Veterinary Medicine, (in Chinese), 12: 78-80  

Cheng D, Sun H, Xu J, Gao S (2005b). Prevalence of Fimbial 
Colonization Factors F18ab and F18ac in Escherichia coli Isolates 
from Weaned Piglets with Edema and/or Diarrhea in China. Vet. 
Microbiol., 110: 35-39.  

Cheng D, Sun H, Xu J, Gao S (2006). PCR detection of virulence factor 
genes in Escherichia coli isolates from weaned piglets with edema 
disease and/or diarrhea in China. Veterinary Vet. Microbiol., 115: 
320-328.  

Fetherston JD, Perry RD (1994). The pigmentation locus of Yersinia 

pestis KIM6
+
 is flanked by an insertion sequence and includes the 

structural genes for pesticin sensitivity and HMWP2. Molecular 
Microbiol., 13: 697-708.  

Guilvout I, Mercereau-Puijalon O. Bonnefoy S, Pugsley AP, Carniel E 
(1993). High-molecular-weight protein 2 of Yersinia enterocolitica is 
homologous to AngR of Vibrio anguillarum and belongs to a family of 
proteins involved in nonribosomal peptide synthesis. J. Bacteriol., 
175: 5488-5504.  

Hacker J, Kaper JB (2000). Pathogenicity islands and the evolution of 
microbes. Ann. Rev. Microbiol., 54: 641-679.  

Karch H, Schubert S, Zhang D, Zhang W, Schmidt H, Olschlager T, 
Hacker J (1999). A genomic island, termed high-pathogenicity island, 



 
 
 

 
is present in certain non-O157 Shiga toxin-producing Escherichia coli 

clonal lineages. Infection Immunity, 67: 5994-6001.  
Levine MM (1987). Escherichia coli that cause diarrhea: enterotoxigenic,  

enteropathogenic, enteroinvasive, enterohemorrhagic, and 
enteroadherent. J. Infect. Dis., 155: 377-389.  

Lucier TS, Fetherston JD, Brubaker RR, Perry RD (1996). Iron uptake 
and iron-repressible polypeptides in Yersinia pestis. Infection 
Immunity, 64: 3023-3031.  

Martins MF, Martinez-Rossi NM, Ferreira A, Brocchi M, Yano T, Castro 
AF, Silveira WD (2000). Pathogenic characteristics of Escherichia coli 
strains isolated from newborn piglets with diarrhea in Brazil. Vet. 
Microbiol., 76: 51-59.  

Oelschlaeger TA, Hacker J (2004). Impact of pathogenicity islands in 
bacterial diagnostics. Acta Pathologica, Microbiologica et 
Immunologica Scandinavica, 112: 930-936. 

 
 
 
 

 
Perry RD, Pendrak ML, Schuetze P (1990). Identification and cloning of 

a hemin storage locus involved in the pigmentation phenotype of 
Yersinia pestis. J. Bacteriol., 172: 5929-5937.  

Rakin A, Noelting C, Schropp P, Heesemann J (2001). Integrative 
module of the high-pathogenicity island of Yersinia. Molecular 
Microbiol., 39: 407-415.  

Schubert S, Rakin A, Karch H, Carniel E, Heesemann J (1998). 
Prevalence of the “high-pathogenicity island” of Yersinia species 
among Escherichia coli strains that is pathogenic to humans. 
Infection Immunity, 66: 480-485.  

Xu JG, Cheng B, Wen X, Cui S, Ye C (2000). High-pathogenicity island 
of Yersinia spp. in Escherichia coli strains isolated from diarrhea 
patients in China. J. Clin. Microbiol., 38: 4672-4675. 


