In vitro assessment of pathogenicity and virulence encoding gene profiles of avian pathogenic Escherichia coli strains associated with colibacillosis in chickens

Document Type : Full paper (Original article)


1 Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, University of Nigeria, Nsukka, Enugu State, Nigeria

2 Department of Bioprocess Technology, School of Industrial Technology, University Sains Malaysia, Penang, Malaysia

3 Department of Animal Science and Technology, School of Agriculture and Agricultural Technology, Federal University of Technology, Owerri, Imo State, Nigeria


Background: Avian pathogenic Escherichia coli (APEC) strains have been associated with various disease conditions in avian species due to virulence attributes associated with the organism. Aims: This study was carried out to determine the in vitro pathogenic characteristics and virulence encoding genes found in E. coli strains associated with colibacillosis in chickens. Methods: Fifty-two stock cultures of E. coli strains isolated from chickens diagnosed of colibacillosis were tested for their ability to produce haemolysis on blood agar and take up Congo red dye. Molecular characterization was carried out by polymerase chain reaction (PCR) amplification of virulence encoding genes associated with APEC. Results: Eleven (22%) and 41 (71%) were positive for haemolysis on 5% sheep red blood agar and Congo red agar, respectively. Nine virulence-associated genes were detected as follows: FimH (96%), csgA (52%), iss (48%), iut (33%), tsh (21%), cva (15%), kpsII (10%), pap (2%), and felA (2%). Conclusion: The APEC strains exhibited virulence properties and harbored virulence encoding genes which could be a threat to the poultry population and public health. The putative virulence genes were diverse and different in almost all isolate implying that pathogenesis was multi-factorial and the infection was multi-faceted which could be a source of concern in the detection and control of APEC infections.


Main Subjects

Al-Arfaj, AA; Ali, MS; Hessain, AM; Zakri, AM; Dawoud, TM; Al-Maary, KS and Moussa, IM (2016). Phenotypic analysis of pathogenic E. coli virulence genes recovered from Riyadh, Saudi Arabia. Saudi J. Biol. Sci., 23: 713-717.
AL-Saiedi, RLR and Al-Mayah, AAS (2014). Pathogenicity testing of several APEC isolates obtained from naturally infected broiler birds reared in Basrah. Int. J. Poult. Sci., 13: 374-378.
Ali, A; Abd El-Mawgoud, AI; Dahshan, AM; EL-Sawah, A and Nasef, SA (2019). Escherichia coli in broiler chickens in Egypt, its virulence traits and vaccination as an intervention strategy. Novel. Res. Microbiol. J., 3: 415-427.
Ambalam, P; Kanthi, KK; Ingrid, N; Torkel, W and Asa, L (2012). Bile stimulates cell surface hydrophobicity, Congo red binding and biofilm formation of Lactobacillus strains. FEMS Microb. Lett., 333: 10-19.
Barbieri, NL; de Oliveira, AL; Tejkowski, TM; Pavanelo, DB; Rocha, DA; Matter, LB; Callegari-Jacques, SM; de Brito, BG and Horn, F (2013). Genotypes and pathogenicity of cellulitis isolates reveal traits that modulate APEC virulence. PLoS One. 8: e72322.
Barbieri, NL; Oliveira, AL; Tejkowski, TM; Pavanelo, DB; Matter, LB; Pinheiro, SRS; Vaz, TMI; Nolan, LK; Logue, CM; Brito, BG and Horn, F (2015). Molecular characterization and clonal relationships among Escherichia coli strains isolated from broiler chickens with colisepticemia. Foodborne Pathog. Dis., 12: 74-83.
Bauchart, P; Pierre, G; Brée, A; Oswald, E; Hacker, J and Dobrindt, U (2010). Pathogenomic comparison of human extraintestinal and avian pathogenic Escherichia coli: Search for factors involved in host specificity or zoonotic potential. Microb. Pathog., 49: 105-115.
Borzi, MM; Cardozo, MV; Oliveira, ES; Pollo, AS; Guastalli, EAL; Santos, LFD and Avila, FA (2018). Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl. Braz. J. Microbiol., 49: 107-112.
Circella, E; Pennelli, D; Tagliabue, S and Camarda, A (2012). Virulence-associated genes in avian pathogenic Escherichia coli from laying hens in Apulia, Southern Italy. Brit. Poult. Sci., 53: 465-470.
Daga, AP; Koga, VL; Soncini, JGM; de Matos, CM; Perugini, MRE; Pelisson, M; Kobayashi, RKT and Vespero, EC (2019). Escherichia coli bloodstream infections in patients at a University hospital: Virulence factors and clinical characteristics. Front. Cell. Infect. Microbiol., 9: 191-200.
Dho-Moulin, M and Fairbrother, JM (1999). Avian pathogenic Escherichia coli (APEC). Vet. Res., 30: 299-316.
Dissanayake, DR; Octavia, S and Lan, R (2014). Population structure and virulence content of avian pathogenic Escherichia coli isolated from outbreaks in Sri Lanka. Vet. Microbiol., 31: 403-412.
Fakruddin, MD; Reaz, MM; Abhijit, C and Khanjada Bin Mannan, S (2012). Comparative analysis of virulence factors of Escherichia coli from non-enteric infections. J. Med. Sci., 12: 37-44.
Garénaux, A; Mélissa, C and Dozois, CM (2011). The Ins and Outs of siderophore mediated iron uptake by extra-intestinal pathogenic Escherichia coli. Vet. Microbiol., 153: 89-98.
Gérard, F; Pradel, N and Wu, LF (2005). Bactericidal activity of colicin V is mediated by an inner membrane protein, SdaC, of Escherichia coli. J. Bacteriol., 187: 1945-1950.
Ghafoor, H; Javed, MT; Saleem, G; Rehman, AU; Murtaza, B; Aslam, MS; Manzoor, F; Abadeen, ZUI; Faizan, SM and Bhutta, MN (2017). Gross and histopathological lesions with reference to selected pathogenicity genes (cvaC, iss, iutA, vat, stx1a, stx2a) of Escherichia coli in cases of early chick mortality. J. Innov. Sci., 3: 1-12.
Guabiraba, R and Schouler, C (2015). Avian colibacillosis: still many black holes. FEMS Microbiol. Lett., 362: 118-125.
Hasani, B; Banani, M; Nouri, A; Goudarzi, H and Akhijahani, M (2017). Detection of three virulence genes and antibiotic resistance profiles in Escherichia coli isolates from commercial broilers with colibacillosis in Tabriz, Iran. Arch. Razi Instit., 72: 1-8.
Hojati, Z; Zamanzad, B; Hashemzadeh, M; Molaie, Rand Gholipour, A (2015). The FimH Gene in Uropathogenic Escherichia coli strains isolated from patients with urinary tract infection. Jundishapur J. Microbiol., 8: e17520.
Ionica, F; Virgilia, P; Ioana, G and Catana, N (2012). Molecular typing of avian pathogenic Escherichia coli (APEC) using multiprimer PCR. Sci. Res. Ess., 7: 2868-2873.
Kumar, D; Bhalerao, A; Gupta, RP and Kumari, M (2013). Pathological identification of minimal predictors of avian pathogenic Escherichia coli virulence for use as a rapid diagnostic tool. J. Clin. Microbiol., 46: 3987-3996.
Mainil, J (2013). Escherichia coli virulence factors. Vet. Immun. Immunopathol., 152: 2-12.
Mbanga, J and Nyararai, YO (2015). Virulence gene profiles of avian pathogenic E. coli isolated from chicken with colibacillosis in Bulawayo, Zimbabwe. Onderstepoort J. Vet. Res., 82: 1-8.
Melican, K; Sandoval, RM; Kader, A; Josefsson, L; Tanner, GA; Molitoris, BA and Richter-Dahlfors, A (2011). Uropathogenic Escherichia coli P and Type 1 fimbriae act in synergy in a living host to facilitate renal colonization leading to nephron obstruction. PLoS Pathog., 7: e1001298.
Mellata, M (2013). Human and avian extraintestinal pathogenic Escherichia coli: infections, zoonotic risks, and antibiotics resistance trends. Foodborne Pathol. Dis., 11: 916-931.
Mohamed, L; Ge, Z; Yuehua, L; Yubin, G; Rachid, K; Mustapha, O; Junwei, Wand Karine, O (2018). Virulence traits of avian pathogenic (APEC) and fecal (AFEC) E. coli isolated from broiler chickens in Algeria. Trop. Ani. Health Product., 50: 547-553.
Mohamed, MA; Shehata, MA and Rafeek, E (2014). Virulence genes content and antimicrobial resistance in Escherichia coli from broiler chickens. Vet. Med. Intl., 2014: 195189. doi:10.1155/2014/195189.
Nakazato, G; Compos, AT; Stehling, EG; Brocchi, M and da Silveira, WD (2009). Virulence factor of avian pathogenic Escherichia coli (APEC). Pesq. Vet. Brasil. 29: 479-486.
Nolan, LK; Barnes, HJ; Vaillancourt, JP; Abdul-Aziz, T and Logue, CM (2013). Colibacillosis. In: Swayne, DE (Ed.), Diseases of poultry. (13th Edn)., Ames, Wiley-Blackwell. PP: 751-806.
Ogura, Y; Ooka, T; Iguchi, A; Toh, H; Asadulghani, M; Oshima, K; Kodama, T; Abe, H; Nakayam, KK; Tobe, T; Hattori, M and Hayashi, T (2009). Comparative genomics reveal the mechanism of the parallel evolution of O157 and non-O157 enterohemorrhagic Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America. 106: 17939-17944.
Olarinmoye, AO; Oludotun, OO; Adekunle, AA; Ntiwunka, UG and Tayo, GO (2013). Antibiograms of avian pathogenic Escherichia coli isolates from commercial layers with colibacillosis in Southwest Nigeria. Malaysian J. Microbiol., 9: 317-325.
Oppong, GO; Rapsinski, GJ; Tursi, SA; Biesecker, SG; Klein-Szanto, AJ; Goulian, M; McCauley, C; Healy, C; Wilson, RP and Tükel, C (2015). Biofilm-associated bacterial amyloids dampen inflammation in the gut: oral treatment with curli fibres reduces the severity of hapten-induced colitis in mice. NPJ Biofilms Microbiomes. 1(2015).
Osman, KM; Mustafa, AM; Elhariri, M and AbdElhamed, GS (2012). Identification of serotypes and virulence markers of Escherichia coli isolated from human stool and urine samples in Egypt. Ind. J. Med. Microbiol., 30: 308-313.
Paixao, AC; Ferreira, AC; Fontes, M; Themudo, P; Albuquerque, T; Soares, MC; Fevereiro, M; Martins, L and Corrêa de Ś, MI (2016). Detection of virulence-associated genes in pathogenic and commensal avian Escherichia coli isolates. Poultry Sci., 95: 1646-1652.
Poole, NM; Green, SI; Rajan, A; Vela, LE; Zeng, XL; Estes, MK and Maresso, AW (2017). Role for FimH in extraintestinal pathogenic Escherichia coli invasion and translocation through the intestinal epithelium. Infect. Immun., 85: e00581-17.
Rahdar, M; Rashki, A; Miri, HR and Ghalehnoo, MR (2015). Detection of pap, sfa, afa, foc and fim adhesion-encoding operons in uropathogenic E. coli isolates collected from patients with urinary tract infections. Jundishapur J. Microbiol., 8: e22647.
Rocha, ACGP; Rocha, SLS; Lima-Rosa, CAV; Souza, GF; Moraes, HLS; Salle, FO; Moraes, LB and Salle, CTP (2008). Genes associated with pathogenicity of avian Escherichia coli (APEC) isolated from respiratory cases of poultry. Pesq. Vet. Bras., 28: 183-186.
Rodriguez-Siek, E; Giddings, W; Doetkott, C; Johnson, J and Nolan, K (2005). Characterizing the APEC pathotype. Vet. Res., 36: 241-256.
Samah, E; Soad, AN and Ahmed, ME (2015). Multidrug resistant bacterial pathogens in eggs collected from backyard chickens. Assiut. Vet. Med. J., 61: 87-103.
Sarowska, J; Futoma-Koloch, B; Jama-Kmiecik, A; Frej-Madrzak, M; Ksiazczyk, M; Bugla-Ploskonska, G and Choroszy-Krol, I (2019). Virulence factors, prevalence and potential transmission of extraintestinal pathogenic Escherichia coli isolated from different sources: recent reports. Gut Pathog., 11: 10-25.
Sauer, MM; Jakob, RP; Eras, J; Baday, S; Eris, D; Navarra, G; Bernèche, S; Ernst, B; Maier, T and Glockshuber, R (2016). Catch-bond mechanism of the bacterial adhesin FimH. Nat. Commun., 7: 10738-10750.
Shankar, TVS; Sharma, A and Grover, YP (2010). Studies on different virulence factors of avian pathogenic Escherichia coli. Haryana Vet., 49: 45-47.
Sharada, R; Krishnappa, G; Raghavan, R; Gowda, RNS and Upendra, H (1999). Haemagglutination and Congo red binding of avian Escherichia coli. Indian J. Comp. Microbiol. Immunol. Infect. Dis., 20: 151-152.
Sharif, H; Javed, MT; Ghafoor, H; Youmis, M; Khan, SU; Rehman, AU; Ashfaq, K; Saleem, G; Mansoor, F; Tariq, N and Rafique, A (2018). Association of pathogenicity genes (cvaC, iss, iutA, Six1A, Six2A and vat) of E. coli with gross, histological lesions of colibacillosis in broilers. Biomed. Lett., 4: 1-13.
Sharpe, N (2005). Recipe for buffers and other laboratory solutions used in electrophoresis, PCR and DNA extraction. DNA Extraction Protocols. In: Lougheed Genetics Laboratory Manual. PP: 1-7.
Stella, AE; De Oliveira, MC; Dias da Silva Fontana, VR; Maluta, RP; Borges, CA and de Ávila, FA (2016). Characterization and antimicrobial resistance patterns of Escherichia coli isolated from feces of healthy broiler chickens. Arq. Instit. Biológ., 83: e0392014.
Stromberg, ZR; Johnson, JR; Fairbrother, JM; Kilbourne, J; Van Goor, A; CurtissIII, R and Mellata, M (2017). Evaluation of Escherichia coli isolates from healthy chickens to determine their potential risk to poultry and human health. PLoS One. 12: e0180599.
Subashchandrabose, S and Mobley, HLT (2015). Virulence and fitness determinants of uropathogenic Escherichia coli. Microbiol. Spectr., 3: 1-20 doi: 10.1128/microbiolspec. UTI-0015-2012.
Tseng, CC; Wang, MC; Lin, WH; Liao, IC; Chen, WC; Teng, CH; Yan, JJ; Wu, AB and Wu, JJ (2018). Role of class II P fimbriae and cytokine response in the pathogenesis of Escherichia coli kidney infection in diabetic mice. J. Microb. Immun. Infect., 52: 492-499.
Ugwu, IC; Ugwu, CC; Chah, KF and Okoye, JOA (2017). Antimicrobial resistance profile of avian pathogenic
Escherichia coli isolated from chicken diagnosed with colibacillosis in Enugu State, Nigeria. IJBPAS., 6: 2162-2174.
Van Gerven, N; Van der Verren, SE; Reiter, DM and Remaut, H (2018). The role of functional amyloids in bacterial virulence. J. Mol. Biol., 430: 3657-3684.
Wang, J; Tang, P; Tan, D; Wang, L; Zhang, S; Qiu, Y; Dong, R; Liu, W; Huang, J; Chen, T; Ren, J; Li, C and Liu, HJ (2015). The pathogenicity of chicken pathogenic Escherichia coli is associated with the numbers and combination patterns of virulence-associated genes. Open J. Vet. Med., 05: 243-254.
Wijetunge, DSS; Gongati, S; DebRoy, C; Kim, KS; Couraud, PO; Romero, IA; Weksler, B and Kariyawasam, S (2015). Characterizing the pathotype of neonatal meningitis causing Escherichia coli (NMEC). BMC Microbiol., 15: 211-225.
Won, G; Moon, B; Oh, I; Matsuda, K; Chaudhari, AA; Hur, J; Eo, S; Yu, I; Lee, Y; Kim, B and Lee, JH (2009). Profiles of virulence-associated of avian pathogenic Escherichia coli isolates from chickens with colibacillosis. Poult. Sci., 46: 260-266.
Wu, G; Ehricht, R; Mafura, M; Stokes, M; Smith, N; Pritchard, GC and Woodward, MJ (2012). Escherichia coli isolates from extraintestinal organs of livestock animals harbor diverse virulence genes and belong to multiple genetic lineages. Vet. Microbiol., 160: 197-206.
Zeinab, MS; Amin, G; Rabie, NS and Zaki, MS (2018). Avian colibacillosis: A review. World Rural Observ., 10: 1-5.
Zhuang, QY; Wang, SC; Li, JP; Liu, D; Liu, S; Jiang, WM and Chen, JM (2014). A clinical survey of common avian infectious diseases in China. Avian Dis., 58: 297-302.