- Email: [email protected]
Prevalence and clonal distribution of avian Escherichia coli isolates harboring increased serum survival (iss) gene
C 2015 Poultry Science Association Inc.
Prevalence and clonal distribution of avian Escherichia coli isolates harboring increased serum survival (iss) gene Mahdi Askari Badouei,∗,1 Patrick Joseph Blackall,† Alireza Koochakzadeh,‡ Hadi Haghbin Nazarpak,# and Mohammad Amin Sepehri§ Department of Pathobiology, Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran; † Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Australia; ‡ Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Iran; # Department of Clinical Sciences, Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran; and § Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran Primary Audience: Poultry Microbiologists, Vaccine Scientists, Agricultural Faculty SUMMARY Colibacillosis, an important disease of poultry caused by avian pathogenic Escherichia coli (APEC), is responsible for great economic losses in the poultry industry around the world. The aims of the present study were to investigate the prevalence of the increased serum survival (iss) gene and to compare the clonal distribution and relatedness of the isolates among septicemic and cecal (commensal) E. coli isolates from poultry in Iran using a random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). A total of 97 (41 septicemic and 56 cecal isolates) were screened for the presence of the iss gene. Then, 66 isolates were randomly chosen for clonal comparison, including 44 iss-positive and 22 iss-negative isolates. The prevalence of the iss gene was 90.3% (37/41) in the septicemic and 64.3% (36/56) among cecal isolates, a significant difference (p = 0.004). The 66 isolates examined by RAPD-PCR were assigned to 4 main clusters, of which 2 were predominant (B and D, 65.6%). As 3 of the 4 clusters, including the 2 largest clusters, did not differ in the relative distribution of iss-positive isolates and as all 4 clusters did not differ in the source of isolates (tissue as compared to cecum), it appears that potentially pathogenic strains can be commonly distributed among the intestinal bacterial flora of birds. The fingerprinting analysis also yielded a high variety of RAPD profiles, indicating a substantial diversity among avian E. coli strains. Key words: APEC, Escherichia coli, iss gene, RAPD-PCR, clonal analysis 2016 J. Appl. Poult. Res. 25:67–73 http://dx.doi.org/10.3382/japr/pfv064
DESCRIPTION OF PROBLEM Escherichia coli is generally considered normal flora of the poultry intestinal tract. Some E. 1
Corresponding author: [email protected]
coli strains, harboring different sets of virulence factors termed avian pathogenic E. coli (APEC), cause the economically important disease known as colibacillosis. A range of conditions in poultry, such as peritonitis, pericarditis, airsacculitis, osteomyelitis, cellulitis, synovitis, salpingitis,
Downloaded from http://japr.oxfordjournals.org/ at Flinders University of South Australia on February 6, 2016
∗
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68
In the present study, the occurrence of the iss gene was first determined in E. coli isolated from cecal contents and tissues (the latter being regarded as linked to colisepticemia) in commercial broilers. Then, the clonal distribution and relatedness of the cecal and septicemic isolates were analyzed using RAPD-PCR method.
MATERIALS AND METHODS Sampling and E. coli Strains The birds were collected from 3 broiler farms (Ross 308) from May to August 2012 and sent for postmortem examination. The selected farms were located in geographically separated regions in the vicinity of Garmsar, Iran. The number of birds in each farm ranged from 15,000 to 25,000. Carcasses were transported to the laboratory in ice buckets and if the carcass condition was not satisfactory it was excluded from the study. A total of 57 carcasses were examined and the sampled birds belonged to different stages of growth, ranging from the third wk of age until close to the final stage of growth (8 wk of age). Tissue samples for isolation were taken at necropsy if lesions suggestive of colibacillosis were present. Specifically, liver and heart blood samples were taken from carcasses with significant pericarditis and perihepatitis. Cecal contents also were sampled from the same carcasses. The samples were cultured on MacConkey agar (Merck, Germany) and after 24 h incubation at 37◦ C, one suspect lactose-fermenting colony was chosen from each plate. After a sub-culture to ensure the purity of the isolates, the strains were characterized by evaluation of biochemical tests, including conventional lactose and glucose fermentation (using TSI medium); production of urease and lysine decarboxylase; the indole, methyl red, and Voges Proskauer tests; and utlilization of citrate [14]. DNA Extraction For DNA extraction, a loopful of each of the confirmed E. coli isolates, from overnight agar culture, was transferred into a 5 mL LB Broth (Merck, Germany). After incubation at 37◦ C for 18 to 20 h, 500 μl culture was transferred into
Downloaded from http://japr.oxfordjournals.org/ at Flinders University of South Australia on February 6, 2016
yolk sac infection, and colisepticemia, is associated with APEC [1, 2]. In general, the various forms of clibacillosis are considered the most common bacterial infections affecting the poultry industry worldwide. Economic losses are due not only to significant morbidity and mortality, but also to a lower average body weight gain and carcass condemnation at processing plants. Poor management practices such as unsatisfactory ventilation, overcrowding, nutritional deficiencies, and concomitant infections with other bacterial or viral agents predispose birds to colibacillosis [1, 3]. The increased serum survival (iss) gene, first described in the ColV plasmid, plays a role in resistance against serum complement [4]. The gene encodes the Iss protein, which has a signal sequence characteristic of outer membrane proteins (OMP) and encodes a 9 to 10 KDa lipoprotein of the bacterial outer membrane [4, 5]. The particular importance of Iss in pathogenesis of APEC strains was first documented when APEC strains with an inactivated iss gene showed reduced pathogenicity in an embryo mortality assay in comparison with the parental wild type strains [6]. Although the accumulative data obtained from many research studies strongly suggest that collibacillosis is attributed to several virulence determinants of APEC, the iss gene is considered one of the most widespread virulence genes in extra-intestinal pathogenic strains [7, 8]. This feature makes the virulence determinant one of the targets for presumptive identification of APEC strains [7– 9], and also a potential candidate for vaccine production [10]. Studying the clonal distribution and diversity among pathogenic and non-pathogenic strains of Escherichia coli may help to explain host species-specificity and expand our knowledge on epidemiology of disease and source of infection [11]. Various techniques are available for bacterial fingerprinting, including random amplified polymorphic DNA (RAPD), enterobacterial repetitive insertion consensus sequences (ERIC) PCR, and pulsed field gel electrophoresis (PFGE) [1, 12, 13]. As RAPD-PCR is a sensitive and practical method, it has been used widely to examine the clonal relationships in epidemiological studies [1, 11].
ASKARI BADOUEI ET AL.: CLONAL DISTRIBUTION OF AVIAN E. COLI sterile microtubes. Samples were centrifuged at 11,000xg for 2 min to sediment bacteria, and the supernatant gently discarded. Then, 250 μl ultrapure water was added and the tube vortexed vigorously for 10 s. Finally, DNA was extracted as described previously and DNA concentrations were adjusted to about 20 ng/μl using a spectrophotometer [15].
The E. coli isolates were subjected to specific PCR for detection of the iss gene using issF: (5 -CAGCAACCCGAACCACTTGATG-3 ) and iss-R: (5 -AGCATTGCCAGAGCGGCAG AA-3 ) primers [7]. The PCR mix contained 1.25 U Taq DNA polymerase (Cinnagen, Iran), 60 ng DNA, 0.7 μM each primer, 200 μM dNTP, and 2 mM MgCl2 , 2.5 μl 10 X PCR buffer and sufficient ultrapure water to achieve a final reaction volume of 25 μl. The PCR program was as follows: initial denaturation at 95◦ C for 4 min and then 30 cycles of denaturation at 94◦ C for 30 s, annealing at 63◦ C for 30 s, and extension at 72◦ C for 1 min. A final extension was conducted at 72◦ C for 5 min. The positive control was a virulent O78: K80 APEC strain while the negative control was sterile water. The presence of the expected 323 base pair (bp) product was recorded as positive [7]. The gels were loaded with a positive and negative control and a 100 bp-plus DNA marker (Vivantis, Malaysia). DNA Fingerprinting by RAPD-PCR For phylogenetic analysis, a total of 66 isolates were randomly chosen from different birds belonging to different farms; 44 were isspositive consisting of 27 colibacillosis and 17 cecal isolates, and 22 were iss-negative consisting of 18 cecal and 4 colibacillosis isolates. The RAPD-PCR was performed as previously described [11]. The PCR mixture included 40 ng DNA, 2.5 mM MgCl2 , 0.5 μM primer (5 -AAGAGCCCGT-3 ), 1.5 U Taq DNA Polymerase (Cinnagen, Iran), 250 μM dNTP mix in 1 X PCR buffer, and ultrapure water up to 25 μl. The PCR conditions were 4 min at 94◦ C for initial denaturation and then 35 cycles of 94◦ C for 45 s, 40◦ C for 40 s, and 72◦ C for
2 min with a 5 min final extension at 72◦ C. The PCR products were electrophoresed at 70 v for 4 h on 2% agarose gels. The RAPD analysis was repeated to ensure the reproducibility of test. The analysis of the RAPD-PCR patterns was carried out by the unweighted pair-group method with arithmetic mean clustering (UPGMA) method using GelQuest (Version 3.1.7) and ClusterVis (version 1.8.1) software. To further examine the possibility that the distribution of potentially pathogenic strains (iss-positive) in each cluster is significant or not, statistical analysis was carried out (2 tailed, Fishers exact test).
RESULTS AND DISSCUSSION Prevalence of iss Gene A total of 97 E. coli were isolated from the examined birds. Of these, 41 were from the affected tissues (septicemic) and 56 were from the cecum (commensal). In total, 73 isolates were shown to harbor the iss gene. The prevalence of the iss gene was 90.3% (37/41) in septicemic and 64.3% (36/56) in cecal isolates. Statistical analysis using Chi-square showed that the presence of the iss gene was significantly higher in the tissue isolates than in the cecal isolates (90.3 vs 64.3%; p = 0.004). Many studies also have shown that the isolation rate of the iss gene in colibacillosis isolates is higher than commensal isolates [16–20]. However, the rates varied remarkably in different studies. Yaguchi et al. (2007) reported a prevalence of iss of 97.6 and 49% in colibacillosis and fecal isolates, respectively [16]. A much lower prevalence was reported by Delicato et al. (2003)—38.5% in colibacillosis isolates and 16% in fecal isolates [2]. Other studies have reported results in between the two cited above, e.g. Pfaff-McDonough et al. [17] reported 78.7 and 18.7%, and RodriguezSiek et al. [7] reported 82.7 and 18.3% for collibacillosis and fecal isolates, respectively. More recently, Kafshdouzan et al. (2013) showed the presence of iss in 68.2% of APEC and 24.5% of fecal isolates in Iran [18]. In conclusion, in agreement with results of the present study, the higher iss prevalence in APEC was repeatedly reported in most previous investigations.
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Detection of iss Gene
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Figure 1. Unweighted pair-group method with arithmetic mean clustering (UPGMA) dendrogram based on data from RAPD-PCR analysis of 66 E. coli isolates. F (Fecal/Cecal), S (Septicemic).
Clonal Analysis of E. coli Strains Analysis of the RAPD-PCR patterns showed that 64 of the 66 examined isolates were classified into 4 main clusters. Two fecal isspositive isolates did not belong to any cluster
and were not included in data analysis of clusters (Figure 1). Cluster B was the main group and 23 isolates (35.9%) were classified into this group. The second major cluster was cluster D with 19 strains (29.7%). Clusters A and C were the minor groups and included only 12 (18.7%) and
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Table 1. Distribution of 64 E. coli isolates in 4 clusters and related sub-clusters as established by RAPD-PCR. Clusters/subclusters
No. of isolates
iss+
iss-
Cecum
Tissue
A B
12 9 14 23 10 9 10 19
9 6 13 19 1 6 7 13
3 3 1 4 9 3 3 6
4 7 7 14 6 6 3 9
8 2 7 9 4 3 7 10
C D
B1 B2 Total D1 D2 Total
Cluster
Presence of iss Positive
Negative
P-value
Origin Tissue
Cecum
P-value
A B C D
9 19 1 13
3 4 9 6
1.0 0.31 0.02∗ 1.0
8 9 4 10
4 14 6 9
0.68 0.76 1.0 1.0
∗
The relative prevalence of iss-positive isolates within cluster C is significantly different (P<0.05) from that seen in the other clusters.
10 (15.6%) isolates, respectively. All the strains in cluster C were iss-negative except one isolate (Table 1). Distribution of iss-harboring and negative strains in each cluster with regard to the origin of the isolates (septicemic vs. cecal) is depicted in Figure 1. In the present study, 64 isolates were grouped into 4 main clusters with 2 clusters (A and B) containing most of the iss-positive isolates. Most of the isolates in cluster A were colisepticemic (8/12), while most isolates in cluster B were of cecal origin (Table 2). Cluster C was the smallest group with only 10 isolates, of which 9 were iss-negative. The majority of isolates in cluster D were iss-positive (13/19), of which about half were associated with colibacillosis (n = 10), and the remaining were cecal isolates (n = 9). However, the statistical analysis of strains in 3 clusters (A, B, D) showed no significant differences over the normal distribution of the iss gene in septicemic and cecal isolates. As well, there was only one group with a significantly lower relative incidence of the iss gene (cluster C). The results of the current study suggest that avian E. coli isolates can be generally classified into a few clonal lineages according to their genotypic differences. More importantly, isspositive (and, therefore, presumably pathogenic) strains are widely dispersed among commensal
E. coli in birds. For instance, in the predominant B cluster, of the 19 iss-positive isolates, 10 were cecal and the remaining 9 were colisepticemic isolates (Figure 1). In addition, it can be concluded that avian cecum contains potentially pathogenic E. coli strains, but the strains with a rich repertoire of virulence determinants have more chance to evade the host defense mechanisms and cause systemic infections. It is worth noting that Collingwood et al. (2014) have recently suggested that APEC is not an appropriate term, because avian pathogenic E. coli strains are more opportunistic pathogens than an easily distinguishable pathotype [21]. A similar marked degree of diversity in RAPD patterns of APEC strains has been reported in other studies [11, 22]. In particular, Chansiripornchai et al. [11] studied 50 APEC strains that yielded 44 RAPD types and Salehi et al. [23] also observed 29 RAPD types in 33 avian E. coli isolates. Although the discrimination index of RAPD was good (0.98 to 0.99) in both studies, the fecal and pathogenic strains still grouped together. A similar finding of great diversity but no distinction between colibacillosis and fecal isolates also has been reported when another typing technique, ERIC-PCR, was used [4, 24, 25, 26]. Notably, the addition of a pathogenicity marker in the present study
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Table 2. Statistical analysis of 64 isolates in the main RAPD-PCR clusters with regard to possession of the iss gene and origin.
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72 (iss gene presence/absence) was not helpful in better clonal discrimination of APEC strains. Based on these results, studies on the clonal relationships of avian E. coli strains that need information on pathogenicity should use confirmatory pathogenicity tests (e.g. the embryo mortality test) and not rely on the source of avian E. coli isolates origin (e.g. fecal as opposed to tissue).
1. The results of the present study indicated a variety of RAPD-PCR profiles in commensal and septicemic isolates that showed the diversity and heterogeneity of avian Escherichia coli. 2. The prevalence of the iss gene was significantly higher in tissue (septicemic) isolates that reflect the importance of this virulence marker in pathogenesis of APEC strains as previously described. 3. The RAPD-PCR was not able to discriminate between fecal and septicemic isolates. Additionally, 3 of the 4 clusters recognized by RAPD-PCR did not differ in the relative prevalence of iss-positive isolates. These findings suggest that potentially pathogenic E. coli strains are distributed among fecal strains and that colibacillosis is an opportunistic infection in nature. The opportunistic nature of this pathogen in poultry can be a challenging factor in developing effective vaccines for colibacillosis.
CONFLICT OF INTEREST The authors declare that they have no conflict of interest.
REFERENCES AND NOTES 1. Nolan, L.K., H.G. Barnes, J.P. Vailancourt, T. AbdulAziz, and C.M. Logue. 2013. Collibacillosis. Pages 751–804 in Disease of poultry. Swayne, D.E., ed. 13th ed. WileyBlackwell, Ames, IA USA. 2. Delicato, E. R., B. G. de Brito, L. C. Gaziri, and M. Vidotto. 2003. Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis. Vet. Microbiol. 94:97–103.
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CONCLUSIONS AND APPLICATIONS
3. Janben, T., C. Schwarz, P. Preikschat, M. Voss, H. C. Philipp, and L. H. Wieler. 2001. Virulence-associated genes in avian pathogenic Escherichia coli (APEC) isolated from internal organs of poultry having died from colibacillosis. Int. J. Med. Microbiol. 291:371–378. 4. Nolan, L. K., S. M. Horne, C. W. Giddings, S. L. Foley, T. J. Johnson, A. M. Lynne, and J. Skyberg. 2003. Resistance to serum complement, iss, and virulence of avian Escherichia coli. Vet. Res. Commun. 27:101–110. 5. Binns, M. M., D. L. Davies, and K. G. Hardy. 1979. Cloned fragments of the plasmid ColV, I-K94 specifying virulence and serum resistance. Nature 279:778–781. 6. Nolan, L. K., R. E Wooley, and R. K. Cooper. 1992. Transposon mutagenesis used to study the role of complement resistance in the virulence of an avian Escherichia coli isolate. Avian Dis. 36:398–402. 7. Rodriguez-Siek, K. E., C. W. Giddings, C. Doetkott, T. G. Johnson, and L. K. Nolan. 2005. Characterizing the APEC pathotype. Vet. Res. 36:241–256. 8. Johnson, T. J., Y. Wannemuehler, C. Doetkott, S. J. Johnson, S. C. Rosenberger, and L. K. Nolan. 2008. Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J. Clin. Microbiol. 46:3987–3996. 9. Derakhshandeh, A., T. Zahraei Salehi, H. Tadjbakhsh, and V. Karimi. 2009. Identification, cloning and sequencing of Escherichia coli strain χ 1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran. Lett. Appl. Microbiol. 49:403–407. 10. Lynne, A. M., S. Kariyawasam, Y. Wannemuehler, T. G. Johnson, S. J. Johnson, A. S. Sinha, D. K. Lynne, H. W. Moon, D. M. Jordan, C. M. Logue, S. L. Foley, and L. K. Nolan. 2012. Recombinant Iss as a potential vaccine for avian colibacillosis. Avian Dis. 56:192–199. 11. Chansiripornchai, N.,Ramasoota, P., J. Sasipreeyajan, and S. B. Svenson. 2000. Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplified polymorphic DNA (RAPD) analysis. Vet. Microbiol. 80:75–83. 12. Welsh, J., and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18:7213–7218. 13. Williams, J. G. K., K. M. Hanafey, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetics marker. Nucleic Acid Res. 18:6531–6535. 14. Markey, B., F. Leonard, M. Archambault, A. Cullinane, and M. D. Maguire. 2013. Clinical Veterinary Microbiology. 2nd ed. Mosby publishing, London. 15. Sambrook, J., and D. W. Russell. 2001. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 16. Yaguchi, K., T. Ogitani, R. Osawa, M. Kawano, N. Kokumai, T. Kaneshige, T. Noro, K. Masubuchi, and Y. Shimizu. 2007. Virulence factors of avian pathogenic Escherichia coli strains isolated from chickens with colisepticemia in Japan. Avian Dis. 51:656–662. 17. Pfaff-McDonough, S. J., S. M. Horne, C. W. Giddings, J. O. Ebert, C. Doetkott, M. H. Smith, and L. K. Nolan. 2000. Complement resistance-related traits among Escherichia coli isolates from apparently healthy birds and birds with colibacillosis. Avian Dis. 44:23–33. 18. Kafshdouzan, K. H., T. Zahraei Salehi, B. Nayeri Fasaei, O. Madadgar, S. H. Yamasaki, A. Hinenoya, and N. Yasuda. 2013. Distribution of virulence associated genes in
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Escherichia coli by RAPD-PCR. Braz. J. Microbiol. 39:494– 497. 24. da Silveira, W. D., A. Ferreira, M. Lancellotti, I. A. Barbosa, D. S. Leite, A. F. de Castro, and M. Brocchi. 2002. Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)–PCR. Vet. Microbiol. 89: 323–328. 25. de Moura, A. C., K. Irino, and M. C. Vidotto. 2001. Genetic variability of avian Escherichia coli isolates evaluated by enterobacterial reptitive intergenic consensus and repetitive extragenic palindromic polymerase chain reaction. Avian Dis. 45:173–181. 26. Soltani, M., S. M. Peighambari, M. Askari Badouei, and A. Sadrzadeh. 2013. Molecular typing of avian Escherichia coli isolates by enterobacterial repetitive intergenic consensus sequences polymerase chain reaction (ERIC-PCR). Iran. J. Vet. Med. 6:143–148.
Acknowledgments The authors would like to thank Islamic Azad University for partial financial support and Dr. Abdollah Derakhshandeh for reviewing this manuscript.
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isolated Escherichia coli from avian colibacillosis. Iran. J. Vet. Med. 7:1–6. 19. Hussein, A. H., I. A. Ghanem, A. A. Eid, M. A. Ali, J. S. Sherwood, G. Li, L. K. Nolan, and C. M. Logue. 2013. Molecular and phenotypic characterization of Escherichia coli isolated from broiler chicken flocks in Egypt. Avian Dis. 57:602–611. 20. Kemmett, K., T. Humphrey, S. Rushton, A. Close, P. Wigley, and N. J. Williams. 2013. A longitudinal study simultaneously exploring the carriage of APEC virulence associated genes and the molecular epidemiology of fecal and systemic E. coli in commercial broiler chickens. Plos One. 8:e67749. doi:10.1371/journal.pone. 0067749. 21. Collingwood, C., K. Kemmett, N. Williams, and P. Wigley. 2014. Is the concept of avian pathogenic Escherichia coli as a single pathotype fundamentally flawed? Front. Vet. Sci. doi:10.3389/fvets.2014.00005. 22. Maurer, J. J., M. D. Lee, C. Lobsinger, T. Brown, M. Maier, and S. G. Thayer. 1998. Molecular typing of avian Escherichia coli isolates by random amplification of polymorphic DNA. Avian Dis. 42:431– 451. 23. Salehi, T. Z., S. A. Madani, V. Karimi, and F. A. Khazaeli. 2008. Molecular genetic differentiation of avian
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Prevalence and clonal distribution of avian Escherichia coli isolates harboring increased serum survival (iss) gene Mahdi Askari Badouei,∗,1 Patrick Joseph Blackall,† Alireza Koochakzadeh,‡ Hadi Haghbin Nazarpak,# and Mohammad Amin Sepehri§ Department of Pathobiology, Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran; † Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Australia; ‡ Department of Microbiology, Faculty of Veterinary Medicine, University of Tehran, Iran; # Department of Clinical Sciences, Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran; and § Faculty of Veterinary Medicine, Garmsar Branch, Islamic Azad University, Garmsar, Iran Primary Audience: Poultry Microbiologists, Vaccine Scientists, Agricultural Faculty SUMMARY Colibacillosis, an important disease of poultry caused by avian pathogenic Escherichia coli (APEC), is responsible for great economic losses in the poultry industry around the world. The aims of the present study were to investigate the prevalence of the increased serum survival (iss) gene and to compare the clonal distribution and relatedness of the isolates among septicemic and cecal (commensal) E. coli isolates from poultry in Iran using a random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). A total of 97 (41 septicemic and 56 cecal isolates) were screened for the presence of the iss gene. Then, 66 isolates were randomly chosen for clonal comparison, including 44 iss-positive and 22 iss-negative isolates. The prevalence of the iss gene was 90.3% (37/41) in the septicemic and 64.3% (36/56) among cecal isolates, a significant difference (p = 0.004). The 66 isolates examined by RAPD-PCR were assigned to 4 main clusters, of which 2 were predominant (B and D, 65.6%). As 3 of the 4 clusters, including the 2 largest clusters, did not differ in the relative distribution of iss-positive isolates and as all 4 clusters did not differ in the source of isolates (tissue as compared to cecum), it appears that potentially pathogenic strains can be commonly distributed among the intestinal bacterial flora of birds. The fingerprinting analysis also yielded a high variety of RAPD profiles, indicating a substantial diversity among avian E. coli strains. Key words: APEC, Escherichia coli, iss gene, RAPD-PCR, clonal analysis 2016 J. Appl. Poult. Res. 25:67–73 http://dx.doi.org/10.3382/japr/pfv064
DESCRIPTION OF PROBLEM Escherichia coli is generally considered normal flora of the poultry intestinal tract. Some E. 1
Corresponding author: [email protected]
coli strains, harboring different sets of virulence factors termed avian pathogenic E. coli (APEC), cause the economically important disease known as colibacillosis. A range of conditions in poultry, such as peritonitis, pericarditis, airsacculitis, osteomyelitis, cellulitis, synovitis, salpingitis,
Downloaded from http://japr.oxfordjournals.org/ at Flinders University of South Australia on February 6, 2016
∗
JAPR: Research Report
68
In the present study, the occurrence of the iss gene was first determined in E. coli isolated from cecal contents and tissues (the latter being regarded as linked to colisepticemia) in commercial broilers. Then, the clonal distribution and relatedness of the cecal and septicemic isolates were analyzed using RAPD-PCR method.
MATERIALS AND METHODS Sampling and E. coli Strains The birds were collected from 3 broiler farms (Ross 308) from May to August 2012 and sent for postmortem examination. The selected farms were located in geographically separated regions in the vicinity of Garmsar, Iran. The number of birds in each farm ranged from 15,000 to 25,000. Carcasses were transported to the laboratory in ice buckets and if the carcass condition was not satisfactory it was excluded from the study. A total of 57 carcasses were examined and the sampled birds belonged to different stages of growth, ranging from the third wk of age until close to the final stage of growth (8 wk of age). Tissue samples for isolation were taken at necropsy if lesions suggestive of colibacillosis were present. Specifically, liver and heart blood samples were taken from carcasses with significant pericarditis and perihepatitis. Cecal contents also were sampled from the same carcasses. The samples were cultured on MacConkey agar (Merck, Germany) and after 24 h incubation at 37◦ C, one suspect lactose-fermenting colony was chosen from each plate. After a sub-culture to ensure the purity of the isolates, the strains were characterized by evaluation of biochemical tests, including conventional lactose and glucose fermentation (using TSI medium); production of urease and lysine decarboxylase; the indole, methyl red, and Voges Proskauer tests; and utlilization of citrate [14]. DNA Extraction For DNA extraction, a loopful of each of the confirmed E. coli isolates, from overnight agar culture, was transferred into a 5 mL LB Broth (Merck, Germany). After incubation at 37◦ C for 18 to 20 h, 500 μl culture was transferred into
Downloaded from http://japr.oxfordjournals.org/ at Flinders University of South Australia on February 6, 2016
yolk sac infection, and colisepticemia, is associated with APEC [1, 2]. In general, the various forms of clibacillosis are considered the most common bacterial infections affecting the poultry industry worldwide. Economic losses are due not only to significant morbidity and mortality, but also to a lower average body weight gain and carcass condemnation at processing plants. Poor management practices such as unsatisfactory ventilation, overcrowding, nutritional deficiencies, and concomitant infections with other bacterial or viral agents predispose birds to colibacillosis [1, 3]. The increased serum survival (iss) gene, first described in the ColV plasmid, plays a role in resistance against serum complement [4]. The gene encodes the Iss protein, which has a signal sequence characteristic of outer membrane proteins (OMP) and encodes a 9 to 10 KDa lipoprotein of the bacterial outer membrane [4, 5]. The particular importance of Iss in pathogenesis of APEC strains was first documented when APEC strains with an inactivated iss gene showed reduced pathogenicity in an embryo mortality assay in comparison with the parental wild type strains [6]. Although the accumulative data obtained from many research studies strongly suggest that collibacillosis is attributed to several virulence determinants of APEC, the iss gene is considered one of the most widespread virulence genes in extra-intestinal pathogenic strains [7, 8]. This feature makes the virulence determinant one of the targets for presumptive identification of APEC strains [7– 9], and also a potential candidate for vaccine production [10]. Studying the clonal distribution and diversity among pathogenic and non-pathogenic strains of Escherichia coli may help to explain host species-specificity and expand our knowledge on epidemiology of disease and source of infection [11]. Various techniques are available for bacterial fingerprinting, including random amplified polymorphic DNA (RAPD), enterobacterial repetitive insertion consensus sequences (ERIC) PCR, and pulsed field gel electrophoresis (PFGE) [1, 12, 13]. As RAPD-PCR is a sensitive and practical method, it has been used widely to examine the clonal relationships in epidemiological studies [1, 11].
ASKARI BADOUEI ET AL.: CLONAL DISTRIBUTION OF AVIAN E. COLI sterile microtubes. Samples were centrifuged at 11,000xg for 2 min to sediment bacteria, and the supernatant gently discarded. Then, 250 μl ultrapure water was added and the tube vortexed vigorously for 10 s. Finally, DNA was extracted as described previously and DNA concentrations were adjusted to about 20 ng/μl using a spectrophotometer [15].
The E. coli isolates were subjected to specific PCR for detection of the iss gene using issF: (5 -CAGCAACCCGAACCACTTGATG-3 ) and iss-R: (5 -AGCATTGCCAGAGCGGCAG AA-3 ) primers [7]. The PCR mix contained 1.25 U Taq DNA polymerase (Cinnagen, Iran), 60 ng DNA, 0.7 μM each primer, 200 μM dNTP, and 2 mM MgCl2 , 2.5 μl 10 X PCR buffer and sufficient ultrapure water to achieve a final reaction volume of 25 μl. The PCR program was as follows: initial denaturation at 95◦ C for 4 min and then 30 cycles of denaturation at 94◦ C for 30 s, annealing at 63◦ C for 30 s, and extension at 72◦ C for 1 min. A final extension was conducted at 72◦ C for 5 min. The positive control was a virulent O78: K80 APEC strain while the negative control was sterile water. The presence of the expected 323 base pair (bp) product was recorded as positive [7]. The gels were loaded with a positive and negative control and a 100 bp-plus DNA marker (Vivantis, Malaysia). DNA Fingerprinting by RAPD-PCR For phylogenetic analysis, a total of 66 isolates were randomly chosen from different birds belonging to different farms; 44 were isspositive consisting of 27 colibacillosis and 17 cecal isolates, and 22 were iss-negative consisting of 18 cecal and 4 colibacillosis isolates. The RAPD-PCR was performed as previously described [11]. The PCR mixture included 40 ng DNA, 2.5 mM MgCl2 , 0.5 μM primer (5 -AAGAGCCCGT-3 ), 1.5 U Taq DNA Polymerase (Cinnagen, Iran), 250 μM dNTP mix in 1 X PCR buffer, and ultrapure water up to 25 μl. The PCR conditions were 4 min at 94◦ C for initial denaturation and then 35 cycles of 94◦ C for 45 s, 40◦ C for 40 s, and 72◦ C for
2 min with a 5 min final extension at 72◦ C. The PCR products were electrophoresed at 70 v for 4 h on 2% agarose gels. The RAPD analysis was repeated to ensure the reproducibility of test. The analysis of the RAPD-PCR patterns was carried out by the unweighted pair-group method with arithmetic mean clustering (UPGMA) method using GelQuest (Version 3.1.7) and ClusterVis (version 1.8.1) software. To further examine the possibility that the distribution of potentially pathogenic strains (iss-positive) in each cluster is significant or not, statistical analysis was carried out (2 tailed, Fishers exact test).
RESULTS AND DISSCUSSION Prevalence of iss Gene A total of 97 E. coli were isolated from the examined birds. Of these, 41 were from the affected tissues (septicemic) and 56 were from the cecum (commensal). In total, 73 isolates were shown to harbor the iss gene. The prevalence of the iss gene was 90.3% (37/41) in septicemic and 64.3% (36/56) in cecal isolates. Statistical analysis using Chi-square showed that the presence of the iss gene was significantly higher in the tissue isolates than in the cecal isolates (90.3 vs 64.3%; p = 0.004). Many studies also have shown that the isolation rate of the iss gene in colibacillosis isolates is higher than commensal isolates [16–20]. However, the rates varied remarkably in different studies. Yaguchi et al. (2007) reported a prevalence of iss of 97.6 and 49% in colibacillosis and fecal isolates, respectively [16]. A much lower prevalence was reported by Delicato et al. (2003)—38.5% in colibacillosis isolates and 16% in fecal isolates [2]. Other studies have reported results in between the two cited above, e.g. Pfaff-McDonough et al. [17] reported 78.7 and 18.7%, and RodriguezSiek et al. [7] reported 82.7 and 18.3% for collibacillosis and fecal isolates, respectively. More recently, Kafshdouzan et al. (2013) showed the presence of iss in 68.2% of APEC and 24.5% of fecal isolates in Iran [18]. In conclusion, in agreement with results of the present study, the higher iss prevalence in APEC was repeatedly reported in most previous investigations.
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Detection of iss Gene
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Figure 1. Unweighted pair-group method with arithmetic mean clustering (UPGMA) dendrogram based on data from RAPD-PCR analysis of 66 E. coli isolates. F (Fecal/Cecal), S (Septicemic).
Clonal Analysis of E. coli Strains Analysis of the RAPD-PCR patterns showed that 64 of the 66 examined isolates were classified into 4 main clusters. Two fecal isspositive isolates did not belong to any cluster
and were not included in data analysis of clusters (Figure 1). Cluster B was the main group and 23 isolates (35.9%) were classified into this group. The second major cluster was cluster D with 19 strains (29.7%). Clusters A and C were the minor groups and included only 12 (18.7%) and
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Table 1. Distribution of 64 E. coli isolates in 4 clusters and related sub-clusters as established by RAPD-PCR. Clusters/subclusters
No. of isolates
iss+
iss-
Cecum
Tissue
A B
12 9 14 23 10 9 10 19
9 6 13 19 1 6 7 13
3 3 1 4 9 3 3 6
4 7 7 14 6 6 3 9
8 2 7 9 4 3 7 10
C D
B1 B2 Total D1 D2 Total
Cluster
Presence of iss Positive
Negative
P-value
Origin Tissue
Cecum
P-value
A B C D
9 19 1 13
3 4 9 6
1.0 0.31 0.02∗ 1.0
8 9 4 10
4 14 6 9
0.68 0.76 1.0 1.0
∗
The relative prevalence of iss-positive isolates within cluster C is significantly different (P<0.05) from that seen in the other clusters.
10 (15.6%) isolates, respectively. All the strains in cluster C were iss-negative except one isolate (Table 1). Distribution of iss-harboring and negative strains in each cluster with regard to the origin of the isolates (septicemic vs. cecal) is depicted in Figure 1. In the present study, 64 isolates were grouped into 4 main clusters with 2 clusters (A and B) containing most of the iss-positive isolates. Most of the isolates in cluster A were colisepticemic (8/12), while most isolates in cluster B were of cecal origin (Table 2). Cluster C was the smallest group with only 10 isolates, of which 9 were iss-negative. The majority of isolates in cluster D were iss-positive (13/19), of which about half were associated with colibacillosis (n = 10), and the remaining were cecal isolates (n = 9). However, the statistical analysis of strains in 3 clusters (A, B, D) showed no significant differences over the normal distribution of the iss gene in septicemic and cecal isolates. As well, there was only one group with a significantly lower relative incidence of the iss gene (cluster C). The results of the current study suggest that avian E. coli isolates can be generally classified into a few clonal lineages according to their genotypic differences. More importantly, isspositive (and, therefore, presumably pathogenic) strains are widely dispersed among commensal
E. coli in birds. For instance, in the predominant B cluster, of the 19 iss-positive isolates, 10 were cecal and the remaining 9 were colisepticemic isolates (Figure 1). In addition, it can be concluded that avian cecum contains potentially pathogenic E. coli strains, but the strains with a rich repertoire of virulence determinants have more chance to evade the host defense mechanisms and cause systemic infections. It is worth noting that Collingwood et al. (2014) have recently suggested that APEC is not an appropriate term, because avian pathogenic E. coli strains are more opportunistic pathogens than an easily distinguishable pathotype [21]. A similar marked degree of diversity in RAPD patterns of APEC strains has been reported in other studies [11, 22]. In particular, Chansiripornchai et al. [11] studied 50 APEC strains that yielded 44 RAPD types and Salehi et al. [23] also observed 29 RAPD types in 33 avian E. coli isolates. Although the discrimination index of RAPD was good (0.98 to 0.99) in both studies, the fecal and pathogenic strains still grouped together. A similar finding of great diversity but no distinction between colibacillosis and fecal isolates also has been reported when another typing technique, ERIC-PCR, was used [4, 24, 25, 26]. Notably, the addition of a pathogenicity marker in the present study
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Table 2. Statistical analysis of 64 isolates in the main RAPD-PCR clusters with regard to possession of the iss gene and origin.
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72 (iss gene presence/absence) was not helpful in better clonal discrimination of APEC strains. Based on these results, studies on the clonal relationships of avian E. coli strains that need information on pathogenicity should use confirmatory pathogenicity tests (e.g. the embryo mortality test) and not rely on the source of avian E. coli isolates origin (e.g. fecal as opposed to tissue).
1. The results of the present study indicated a variety of RAPD-PCR profiles in commensal and septicemic isolates that showed the diversity and heterogeneity of avian Escherichia coli. 2. The prevalence of the iss gene was significantly higher in tissue (septicemic) isolates that reflect the importance of this virulence marker in pathogenesis of APEC strains as previously described. 3. The RAPD-PCR was not able to discriminate between fecal and septicemic isolates. Additionally, 3 of the 4 clusters recognized by RAPD-PCR did not differ in the relative prevalence of iss-positive isolates. These findings suggest that potentially pathogenic E. coli strains are distributed among fecal strains and that colibacillosis is an opportunistic infection in nature. The opportunistic nature of this pathogen in poultry can be a challenging factor in developing effective vaccines for colibacillosis.
CONFLICT OF INTEREST The authors declare that they have no conflict of interest.
REFERENCES AND NOTES 1. Nolan, L.K., H.G. Barnes, J.P. Vailancourt, T. AbdulAziz, and C.M. Logue. 2013. Collibacillosis. Pages 751–804 in Disease of poultry. Swayne, D.E., ed. 13th ed. WileyBlackwell, Ames, IA USA. 2. Delicato, E. R., B. G. de Brito, L. C. Gaziri, and M. Vidotto. 2003. Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis. Vet. Microbiol. 94:97–103.
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CONCLUSIONS AND APPLICATIONS
3. Janben, T., C. Schwarz, P. Preikschat, M. Voss, H. C. Philipp, and L. H. Wieler. 2001. Virulence-associated genes in avian pathogenic Escherichia coli (APEC) isolated from internal organs of poultry having died from colibacillosis. Int. J. Med. Microbiol. 291:371–378. 4. Nolan, L. K., S. M. Horne, C. W. Giddings, S. L. Foley, T. J. Johnson, A. M. Lynne, and J. Skyberg. 2003. Resistance to serum complement, iss, and virulence of avian Escherichia coli. Vet. Res. Commun. 27:101–110. 5. Binns, M. M., D. L. Davies, and K. G. Hardy. 1979. Cloned fragments of the plasmid ColV, I-K94 specifying virulence and serum resistance. Nature 279:778–781. 6. Nolan, L. K., R. E Wooley, and R. K. Cooper. 1992. Transposon mutagenesis used to study the role of complement resistance in the virulence of an avian Escherichia coli isolate. Avian Dis. 36:398–402. 7. Rodriguez-Siek, K. E., C. W. Giddings, C. Doetkott, T. G. Johnson, and L. K. Nolan. 2005. Characterizing the APEC pathotype. Vet. Res. 36:241–256. 8. Johnson, T. J., Y. Wannemuehler, C. Doetkott, S. J. Johnson, S. C. Rosenberger, and L. K. Nolan. 2008. Identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J. Clin. Microbiol. 46:3987–3996. 9. Derakhshandeh, A., T. Zahraei Salehi, H. Tadjbakhsh, and V. Karimi. 2009. Identification, cloning and sequencing of Escherichia coli strain χ 1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran. Lett. Appl. Microbiol. 49:403–407. 10. Lynne, A. M., S. Kariyawasam, Y. Wannemuehler, T. G. Johnson, S. J. Johnson, A. S. Sinha, D. K. Lynne, H. W. Moon, D. M. Jordan, C. M. Logue, S. L. Foley, and L. K. Nolan. 2012. Recombinant Iss as a potential vaccine for avian colibacillosis. Avian Dis. 56:192–199. 11. Chansiripornchai, N.,Ramasoota, P., J. Sasipreeyajan, and S. B. Svenson. 2000. Differentiation of avian pathogenic Escherichia coli (APEC) strains by random amplified polymorphic DNA (RAPD) analysis. Vet. Microbiol. 80:75–83. 12. Welsh, J., and M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18:7213–7218. 13. Williams, J. G. K., K. M. Hanafey, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetics marker. Nucleic Acid Res. 18:6531–6535. 14. Markey, B., F. Leonard, M. Archambault, A. Cullinane, and M. D. Maguire. 2013. Clinical Veterinary Microbiology. 2nd ed. Mosby publishing, London. 15. Sambrook, J., and D. W. Russell. 2001. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 16. Yaguchi, K., T. Ogitani, R. Osawa, M. Kawano, N. Kokumai, T. Kaneshige, T. Noro, K. Masubuchi, and Y. Shimizu. 2007. Virulence factors of avian pathogenic Escherichia coli strains isolated from chickens with colisepticemia in Japan. Avian Dis. 51:656–662. 17. Pfaff-McDonough, S. J., S. M. Horne, C. W. Giddings, J. O. Ebert, C. Doetkott, M. H. Smith, and L. K. Nolan. 2000. Complement resistance-related traits among Escherichia coli isolates from apparently healthy birds and birds with colibacillosis. Avian Dis. 44:23–33. 18. Kafshdouzan, K. H., T. Zahraei Salehi, B. Nayeri Fasaei, O. Madadgar, S. H. Yamasaki, A. Hinenoya, and N. Yasuda. 2013. Distribution of virulence associated genes in
ASKARI BADOUEI ET AL.: CLONAL DISTRIBUTION OF AVIAN E. COLI
Escherichia coli by RAPD-PCR. Braz. J. Microbiol. 39:494– 497. 24. da Silveira, W. D., A. Ferreira, M. Lancellotti, I. A. Barbosa, D. S. Leite, A. F. de Castro, and M. Brocchi. 2002. Clonal relationships among avian Escherichia coli isolates determined by enterobacterial repetitive intergenic consensus (ERIC)–PCR. Vet. Microbiol. 89: 323–328. 25. de Moura, A. C., K. Irino, and M. C. Vidotto. 2001. Genetic variability of avian Escherichia coli isolates evaluated by enterobacterial reptitive intergenic consensus and repetitive extragenic palindromic polymerase chain reaction. Avian Dis. 45:173–181. 26. Soltani, M., S. M. Peighambari, M. Askari Badouei, and A. Sadrzadeh. 2013. Molecular typing of avian Escherichia coli isolates by enterobacterial repetitive intergenic consensus sequences polymerase chain reaction (ERIC-PCR). Iran. J. Vet. Med. 6:143–148.
Acknowledgments The authors would like to thank Islamic Azad University for partial financial support and Dr. Abdollah Derakhshandeh for reviewing this manuscript.
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isolated Escherichia coli from avian colibacillosis. Iran. J. Vet. Med. 7:1–6. 19. Hussein, A. H., I. A. Ghanem, A. A. Eid, M. A. Ali, J. S. Sherwood, G. Li, L. K. Nolan, and C. M. Logue. 2013. Molecular and phenotypic characterization of Escherichia coli isolated from broiler chicken flocks in Egypt. Avian Dis. 57:602–611. 20. Kemmett, K., T. Humphrey, S. Rushton, A. Close, P. Wigley, and N. J. Williams. 2013. A longitudinal study simultaneously exploring the carriage of APEC virulence associated genes and the molecular epidemiology of fecal and systemic E. coli in commercial broiler chickens. Plos One. 8:e67749. doi:10.1371/journal.pone. 0067749. 21. Collingwood, C., K. Kemmett, N. Williams, and P. Wigley. 2014. Is the concept of avian pathogenic Escherichia coli as a single pathotype fundamentally flawed? Front. Vet. Sci. doi:10.3389/fvets.2014.00005. 22. Maurer, J. J., M. D. Lee, C. Lobsinger, T. Brown, M. Maier, and S. G. Thayer. 1998. Molecular typing of avian Escherichia coli isolates by random amplification of polymorphic DNA. Avian Dis. 42:431– 451. 23. Salehi, T. Z., S. A. Madani, V. Karimi, and F. A. Khazaeli. 2008. Molecular genetic differentiation of avian
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