Tạp chí Công nghệ Sinh học 11(4): 665-672, 2013
DISTRIBUTION OF SEROGROUPS AND VIRULENCE GENES OF E. COLI STRAINS ISOLATED FROM PORCINE POST WEANING DIARRHEA IN THUA THIEN HUE PROVINCE VIETNAM Nguyen Xuan Hoa1, Dildar Hussain Kalhoro2, Chengping Lu2 1 2
Hue University College of Agriculture and Forestry, Vietnam Nanjing Agricultural University, College of Veterinary Medicine, China SUMMARY Heat-stable (ST) and heat-labile (LT)enterotoxins produced by enterotoxigenic Escherichia coli(E. coli)(ETEC) and Shiga-toxin producing E. coli(STEC) have been as the important factors associated with pathogens to porcine post weaning diarrhea. Ninety fiveE. coli strains were isolated from feces of porcine post weaning diarrhea in Central Vietnam. For serotype, toxins and adhesion encoding genes of the E. coli strains were amplified by polymerase chain reaction (PCR) technique. The results showed that the isolated E. coli strains belong to 9 antisera. In which 76 strains (80.85%) were O-serogroup including O2, O8, O138, O139, O141, O147, O149 and O157, and the others (19 strains accounted 20%) were O-non typeable. Predominant serogroup was O8. Adhesion factor genes detected were 22.11% isolates carried genes for F18, while genes for F4 (K88), F6 (P987) and F5 (K99) were 15.79, 5.26 and 4.21%, respectively. The genes for STa, STb, LT, Stx2e, Stx2a and Stx2l toxins were identified 49.57, 22.14, 13.68, 6.3, 3.16 and 3.16%, respectively. The serogroups were O139/F4/STa/STb/Stx2e (1 isolated), and O45/F4/STa/LT/Stx2e (1 isolate). The hemolytic activity on blood agar plates were detected in 62 isolates (65.26%). Most of E. coli isolates carried genes for adhesions and toxins. Furthermore, our results suggested that there is high genetic variation among E. coli isolates mainly in ETEC and O8 serotype. Keywords: E. coli, serogroup, virulence genes, enterotoxin and shigatoxin Abbreviations: ETEC: enterotoxigenicE. coli; LT: heat-labile; STEC: shiga-toxin producing E. coli. ST: Heatstable; PCR: polymerase chain reaction; PBS: Phosphate buffered saline; PWD: post weaning diarrhea
INTRODUCTION The pig is the most important animal for livestock production in Vietnam, where has been estimated that than 70% of farmer households raise pigs with an average herd side of 5-6 pigs per household (GSO, 2011). Currently, about 74.3% of the meat production is pork, while the share of poultry meat is about 16% and beef and buffalo is about 12.11% (GSO, 2011). In Central Vietnam about 95% of the farmers raise livestock and pig production in this area not only plays an important role provides meat for human, but also provid pig manure for crop production and act as a form of saving and investment (Hong, 2008). However, pig production in Vietnam still has many problems, especially diarrhea is common at post-weaning (Tuyen et al., 2005). Diarrhea caused by E. coli was as a disease associated with industrialized pig production in Asia (Ranald, 2000). Porcine post
weaning diarrhea occurs sporadically or as major outbreaks resulting in economic losses of porcine industry (Kolotilin et al., 2012; Rossi et al., 2012). Post weaning diarrhea (PWD) is caused by a group of bacteria called ETEC and STEC in piglets (Asami et al., 1997; Nguyen et al., 2009). ETEC can cause severe diarrhea in newborns (Wada et al., 1996). PWDis another most constant disease problem among piglets weaned at 3 to 4 weeks of age at large-scale farms that cause diseases through the specified adhesion factors (F4, F5, F6, F18 and F41), fimbriae (Lyutskanov, 2011; Patricia et al., 1990), production of heat-labile enterotoxins and heat-stable enterotoxins (STa or STb) (Amezcua et al., 2002; DebRoy and Maddox, 2001; Wieler et al., 2001). Porcine pathogenic E. coli involved in PWD typically belong to serogroups O2, O8, O138, O139, O141, O147, O149 and O157 (Fairbrother et al., 2005; Han et al., 2007; Meno and Fujimoto, 2002; Silva et al., 2001). PWD is usually the most 665
Nguyễn Xuân Hòa et al. constant problem of large-scale farms and is responsible for significant losses worldwide (Hampson, 1994). Newborn piglets may be protected by antibodies passed through dam milk leading to low incidence of diseases at the early stages of growth (Makino et al., 2001; Wieler et al., 2001). However, after weaning, loss of protective antibodies in milk, transportation, mixing and overcrowding of piglets cause diarrhea (Frydendahl, 2002). Porcine PWD is frequently observed in Central Vietnam, there is a lack of information about the prevalence of serogroups and virulence factors of porcine pathogenic E. coli. The objectives of this study were to determine the serogroups and the virulence genes in E. coli strains isolated from porcine with PWD. MATERIAL AND METHODS Bacterial strains: Ninety five E. coli strains were isolated from fecal samples of 105 porcine PWD in ThueThien Hue province, Central Vietnam. All specimens were obtained within 24 h of the onset of illness and before any antimicrobial treatment to ensure optimum recovery of E. coli. Fecal samples were collected with swabs and transferred into PBS buffer (137 mMNaCl, 2.7 mMKCl and 10 mM Na2HPO4) at 4°C to the laboratory. The samples were streaked on MacConkey agar, incubated at 37°C for 24 h, and then examined for the presence of bacterial colonies. Putative E. coli colonies were identified by morphological and biochemical characteristics, including indole production test, Voges–Proskauer test, citrate utilization, glucose and lactose fermentation, hydrogen sulfate production, and β-hemolysis. All isolates were store at -20°C in Luria-Bertani broth containing 20% glycerol until they were used. The isolates were tested for the presence of serogroups and virulence factors. An E. coli isolate was classified as a strain of ETEC if it carried any of the three enterotoxins virulence genes detected by PCR assay. Extraction of total DNA of E. coli Isolated E. coli strains were inoculated on LB, incubated at 37°C for 18 h. 500 µl of culture was dispensed into 1.5 ml, heat shock at 100°C for 5 mins and put on ice immediately. Centrifuge at 5000 rpm/min for 5 mins. 200 µl of supernatant was taken as DNA template for PCR (Li et al., 2010; Wenz et al., 2006).
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PCR assay PCR was used to detect the toxin (STa, STb, LT and Stx2e) genes, serogroups (O2, O8, O138, O139, O141, O147, O149 and O157) genes, and adhesion (F4, F5, F6, F18 and F41) genes. The sequence of primers and the predicted sizes of amplified product were shown in Table 1. For amplification, PCR was carried out in a total volume of 25 µl containing 1µl of temple DNA, 20µl of each primer, 12.5 µl premix Taq, 9.5µl H 2O. PCR programme consisted of 30 cycles of 94°C for 1 min, annealing temperature specific for each pair of primers for 1 min (Table 1) and 72°C for 2 min in a thermal cycler (Han et al., 2007; Wenz et al., 2006). The amplified DNA was visualized in 1% agarose gel stained with ethidium bromide. The 100 bp ladder was used as a standard. The multiplex PCR primer sets were divided into two groups. The first group targets the O-antigen-specific genes and the second group targets thevirulence genes. RESULTS Isolation and serogroups: In this study, 105 isolates fecal samples, ninety five (90.48%) were E. coli. The E. coliwere positive in serogroups with 9 antisera, 76 strains (80.85%) were O-serogroups and belonged to O2, O8, O138, O139, O141, O147, O149 and O157, while 19 strains (20%) were O-non typeable. The PCR analysis detected the genes for O8 in 23 strains (24.21%), was the most prevalent gene for O serogroups, while the genes for O2, O45, O138, O139, O141, O147, O149, and O157 were found in 4.21, 5.26, 9.47, 6.32, 7.37, 6.32, 9.47 and 7.37% of the isolates, respectively (Table 2 and Figure 1). Toxin and adhesion genes The prevalence of virulence factor genes among the 78 strains isolates from 95 strains PWD E. coli (Table 3 and Figures 2, 3). The majority of the isolates (82.1%) carried multiple genes. The STa gene (49.47%) was most common, followed by STb (21.05), LT (13.68%), Stx2e (6.32%), Stx2a (3.16%), and Stx2l (3.16%). Fimbrial genes were detected in 45 isolates (47.37%), with F18 was the most common (22.11%), followed by F4 (15.79%), F6 (5.26%) and F5 (4.21%). One of the isolates contained two fimbrial genes (F6/F18).
Tạp chí Công nghệ Sinh học 11(4): 665-672, 2013 Table 1. Oligonucleotide sequences, annealing temperature and predicted size of amplification products for different virulence associated genes Target gene
Oligonucleotide sequences (5´- 3´)
Size
*
O2
(F)AGTGAGTTACTTTTTAGCGATGGAC
770
55
O2
(R)AGTTTAGTATGCCCCTGACTTTGAA
770
55
orf469 (O8)
(F)8124-TGTTCGTGCGATGGACC-8140
1,185
55.4
orf469 (O8)
(R)9308-TATCCGAATGGGCCTTCT-9291
1,185
55.1
wzy(O45)
(F) 9401-TGCTTCAATTTGGCTGTT-9418
842
51.8
wzy(O45)
(R)10242-CGTTGGCATTATCGTCTA-10225
842
49.6
wzy(O138)
(F)11536-TGCCGACAACATTATCAA-11553
993
50.3
wzy(O138)
(R)12528-CAAACTTTACCCGACGAA-12511
993
51.5
wzy(O139)
(F) 5003-ATAACGCATCCGCCAACT-5020
1,037
56.1
wzy(O139)
(R) 6039-CCGACTAATACGGAAACA-6022
1,037
48.7
wzy(O141)
(F) 8097-TGAACCTGGGTTTACATT-8114
746
47.5
wzy(O141)
(R)8842-GTACAATTATCATTGCGAGT-8823
746
47.6
wzy(O147)
(F) 6208-TTTTGCTCTTATGGAACC-6225
689
47.9
wzy(O147)
(R) 6896-ATAACGCCAAGTTGATTT-6879
689
47.6
wzy(O149)
(F) 5755-TTTGGTGCAGATACTCAGA-5773
709
48.6
wzy(O149)
(R)6463-GAACAATAGATGCGATACAA-6444
709
48.5
wzy(O157)
(F) 845-TCAGCGGCTAAGTTGATT-862
861
51.2
wzy(O157)
(R) 1705-ATTTGCTCCCATGTCTCC-1688
861
52.1
F4
(F) 169-TGATTTCAATGGTTCGGTC-187
770
53.4
F4
(R) 938-TTGCTACGTTCAGCGGAG-921
770
55.2
F5
(F) 235-CAGGCTGCTATTAGTGGT-252
356
47.7
F5
(R) 590-GCTGAAGTAGTAAATACGC-572
356
45.2
F6
(F) 378-AGTTACTGCCAGTCTATGC-396
396
47
F6
(R) 773-TTTGTATCAGGATTCCCT-756
396
46.6
F18
(F) 66-GCAAGGGGATGTTAAATTC-84
447
52.2
F18
(R) 512-TTGTAAGTAACCGCGTAAG-494
447
49.3
F41
(F) 185-TGACATTATTATTGGTGGTG-204
592
48.7
F41
(R) 776-GTGACTGAGGTCATCCC-760
592
49.9
Stxl
F- CGC TGA ATG TCA TTC GCT CTG C
302
51.6
Stxl
R-CGT GGT ATA GCT ACT GTC ACC
302
49.2
Stx2a
F-CTT CGG TAT CCT ATT CCC GG
516
48.7
Stx2a
R-CTG CTG TGA CAG TGA CAA AAC GC
516
51.9
Stx2e
F-ATG AAG AAG ATG TTT ATA GCG
264
41.4
Stx2e
R-TCA GTT AAA CTT CAC CTG GGC
264
47.3
STa
F-TCC GTG AAA CAA CAT GAC GG
244
46.6
STa
R-ATA ACA TCC AGC ACA GGC AG
244
46.6
STb
F-GCC TAT GCA TCT ACA CAA TC
279
44.6
STb
R-TGA GAA ATG GAC AAT GTC CG
279
44.6
LT
F-ATT TAC GGC GTT ACT ATC CTC
281
45.3
LT
R- CCC CCA AAA TAA TAA GGT ATG
281
43.3
(˚C)
Reference (Li et al., 2010)
(Han et al., 2007)
*
Annealing temperature, F: forward primer, R: reverse primer
667
Nguyễn Xuân Hòa et al. A
B
Figure 1. PCR products amplified from E. coli belonging to O serogroups. Lanes 1-9: O2 (770 bps), O147 (470 bps), O141 (746 bps), O45 (842 bps), O149 (709 bps), O8 (1185 bps), O138 (993 bp), O139 (1037 bps) and O157 (760 bps) respectively.
Table 2. Frequency of O serogroups genes from isolated E. coli strains. O serogroups
Frequency (%)
O2
4.21
O8
24.21
O45
5.26
O138
9.47
O139
6.32
O141
7.37
O147
6.32
O149
9.47
O157
7.37
None detected
20
Table 3. Frequency of individual virulence genes from the isolated E. coli strains Virulence factor
Frequency (%)
F4
15.79
F5
4.21
F6
5.26
F18
22.11
STa
49.47
STb
21.05
LT
13.68
Stx2e
6.32
Stx2a
3.16
Stx2l
3.16
None detected
17.89
668
Tạp chí Công nghệ Sinh học 11(4): 665-672, 2013
Figure 2. PCR produced of fimbrial genes amplification. M: 5000bp Marker, Land 1, 2, 3 are possitive F4 (770 bps), F6 (396 bps) and F18 (447 bps).
Figure 3. PCR products of STa genes amplification. M: 5000 bps Marker, Land 1- 22 E. coli samples. Table 4.Virulence, serogroups genes and hemolytic of the 95 isolated E. coli strains. Virulence genes
Total no. of isolated
Virulence genes
Total no. of isolated
Serogroup(No. of haemolytic/Total no. of isolates)
F4/STb/LT
1
F6/STa
1
O2(4/4)
F4/LT/Stx2e
1
F6/F18
1
O8(16/23)
F4/STa/Stx2e
1
F18/STa/STb
1
O45(5/5)
F4/STb
2
F18/STa
6
O138(8/9)
F4/Stx2e
1
F18/STa/Stx2l
1
O139(5/6)
F4/STa
2
F18/LT
1
O141(6/9)
F4/STa/LT/Stx2e
1
F18/STb
3
O147(6/7)
F4/STa/Stx2l
1
F18
8
O149(5/6)
F4/STa/Stx2a
1
STa/Stx2a
1
O157(7/7)
F4/STa/STb/Stx2e
1
STa/STb
8
F4
1
STa/LT/Stx
1
F4/LT
2
STb/Stx2e
1
F5/STa/LT
1
STa
17
F5/STa
1
LT
3
F5
2
Stx2l
1
F6/STa/LT
1
STb
3
F6/STa/Stx2e
1
None
17
None detected O (2/19)
669
Nguyễn Xuân Hòa et al. Virulence, serogroups genes and hemolytic activity The Table 4 showed that 33 types of virulence factor patterns belonged to the different virulence genes. The combination of most frequent virulence genes were STa, F18, STb, F4, LT, Stx2e, F6, F5, Stx2a and Stx2l. The hemolytic activity on blood agar plates were detected in 62 (65.26%) of the 95 E. coli isolates.Serogroups four genes were O139/F4/STa/STb/Stx2e(1 isolated), O45/F4/STa/LT/Stx2e(1 isolated). DISCUSSION In the present study although 95 isolates belonged to 9 different O serogroups main ETEC and STEC belonged to only three serogroups O8, O138 and O141. O8 (24%) was the most prevalent in, these observations is similar with E. coli isolated from Guangdong province in China (Wang et al., 2011).The presence of ETEC, STEC virulence factors, fimbriae are common features of isolates associated with porcine PWD (Ojeniyi et al., 1994; Vu et al., 2006). The most noteworthy finding of this study was the relatively large numbers of ETEC isolates origin 80 (84.21%), and STEC 12 (12.63%)of genes in E. coli isolates from porcine PWD in the ThuaThien Hue province, and the most prevalent association of the genes was STa, F18, STb, F4, LT, Stx2e, F6, F5, Stx2a and Stx2l.It this study, 82.1% isolates carried multiple virulence genes.F4 and F18 are the most important fimbriae adhesions of ETEC and STEC causing PWD (Hur et al., 2012; Nagy et al., 1997). In the present study, we isolated F4 (15.79%), and F18 (22.11%) genes in E. coli strains that cause PWD inpiglets. However, found F4 (10%) and F18 (35%) genes from pathogenic E. coli isolated from PWD in Republic of Slovak (Vu et al., 2006). Our findings are in accordance with the previous studies of E. coli strains in domestic animals, chickens and pigs (Frydendahl, 2002).Study also reported that E. coli F18ab-positive strains mostly express Shiga toxin while F18ac-positive strains generally responsible for enterotoxins in PWD in pigs. In our present study we first time reported that both variant types may carry genes for Shiga toxins as well as enterotoxins.F18 and STEC variants may be involved in causing of PWD or edema in pigs (Pittman, 2010).The results of the PCR analysis showed the genes for LT or/and ST (STa or/and STb) enterotoxins in E. coli strains isolated from PWD in pigs. The gene for STa was the most 670
prevalent containing (49.47%) while genes for STb and LT were found in 21.05% and 13.68%, respectively. In the previous study of Vidotto(2009) found high frequency in 40, 47 and 71% for STa, STb and LT, respectively.It is well known that porcine STEC produce the edema verotoxin(VTe), also named shiga toxin 2e (Stx2e), which causes damage to the vascular endothelium of the small intestine, subcutaneous edema and neurological disorders. In the present study, frequency of strains (12.63%) were Stx(Stx2e, Stx2a, Stx2l) positive, as compared to the strains isolated in the Slovakia that presented 5% frequency (Vu et al., 2006). HaemolyticE. coli were frequently isolated from faecal samples which had been collected after 5-7 days of weaning but seldom from samples from the same pigs collected before weaning (Svendsen et al., 1977). Although, haemolysis does not seen to play an essential role in the virulence of porcine ETEC and STEC, most of the typical PWD E. coli are hemolytic(Bielaszewska et al., 2011). In the present study 65.26% of isolated were hemolytic. Previous results were also found 71% haemolytic isolates in E. coli strains collected from PWD in piglets in the Republic of Slovakia (Vu et al., 2006). CONCLUSION The present study shows the frequency of serogroups and virulence genes in E. coli strains isolated from porcine PWD in the ThuaThien Hue province, Central Vietnam and also confirm the combination of various virulent genes for ETEC and STEC. Serogroups and virulence genes in E. coli strains are potential nominees for vaccination, bioproductive preventive measures and therapeutic strategies against diarrhea in porcine PWD. Acknowledgments: This study was supported by a grant from the Program for New Century Excellent in University of Ministry of Education of China (NCET-110671), and the Priority Academic Program Development of Jiangsu Higher Education Institutions. REFERENCES Amezcua R, Friendship Robert M, Dewey CE, Gyles C, Fairbrother JM (2002) Presentation of postweaning Escherichia coli diarrhea in southern Ontario, prevalence of haemolytic E. coli serogroups involved, and their antimicrobial resistance patterns. Can J Vet Res 66: 73-78.
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SỰ PHÂN BỐ CỦA NHÓM HUYẾT THANH VÀ ĐỘC LỰC CỦA VI KHUẨN E. COLI PHÂN LẬP TỪ LỢN CON SAU CAI SỮA BỊ BỆNH TIÊU CHẢY TRÊN ĐỊA BÀN TỈNH THỪA THIÊN HUẾ, VIỆT NAM NguyễnXuân Hòa1,*,Dildar Hussain Kalhoro2, Chengping Lu2 1 2
Trường Đại học Nông Lâm, Đại học Huế, Việt Nam Trường Đại học Nông nghiệp Nam Kinh, ViệnThú Y, Trung Quốc TÓM TẮT Tổng số 95 chủng vi khuẩn E. coli đã được phân lập từ phân của lợn con sau cai sữa bị bệnh tiêu chảy trên địa bàn Tỉnh Thừa Thiên Huế. Tất cả các chủng vi khuẩn này được kiểm tra gen qui định nhóm huyết thanh học, sản sinh độc tố và yếu tố bám dính bằng kỹ thuật PCR. Vi khuẩn E. coli phân lập chủ yếu thuộc một trong 9 nhóm huyết thanh như: O2, O8, O138, O139, O141, O147, O149 và O157 chiếm tỷ lệ 80.85% (76 chủng) và 20% không thuộc nhóm huyết thanh này. Nhóm huyết thanh O8 được phát hiện với tần số cao nhất 24.21%. Yếu tố bám dính F18 được phát hiện tương đối cao 22.11% trong khi F4 (K88), F6 (P987) và F5 (K99) theo thứ tự 15.79, 5.26 và 4.21%. Gen mã hóa độc tố đường ruột và độc tố STa, STb, LT tương ứng 49.57, 22.14, 13.68. Gen mã hóa độc tố Shiga, Stx2e, Stx2a và Stx2l theo thứ tư 6.3%, 3.16 3.16%. Chúng tôi đã tìm thấy hai chủng vi khuẩn E. coli hội tụ năm yếu tố độc lực O139/F4/STa/STb/Stx2e và O45/F4/STa/LT/Stx2e. Tần suất của các chủng vi khuẩn E. coli dung huyết phân lập được chiếm 65.26% (62 chủng). Hầu hết các chủng E. coli phân lập được đều mang gen mã hóa yếu tố bám dính và yếu tố độc lực. Nghiên cứu của chúng tôi cho thấy các chủng vi khuẩn E. coli phân lập từ Thừa Thiên Huế chủ yếu thuộc nhóm huyết thanh O8 ETEC. Những chủng này tiềm năng cho các nghiên cứu về thực khuẩn thể, vaccine và chế phẩm sinh học phòng trị bệnh tiêu chảy ở lợn con sau cai sữa. Từ khóa: E. coli, Nhóm huyết thanh, Gen độc, Độc tố đường ruột và Độc tố Shiga
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