Genes associated with pathogenicity of avian Escherichia coli (APEC) isolated from respiratory cases of poultry

Genes associados à patogenicidade de Escherichia coli patogênica para aves (APEC) isoladas de frangos de corte com sintomatologia clínica respiratória

Genes associated with pathogenicity of avian Escherichia coli (APEC) isolated from respiratory cases of poultry

Genes associados à patogenicidade de Escherichia coli patogênica para aves (APEC) isoladas de frangos de corte com sintomatologia clínica respiratória

Ana C.G.P. RochaI, II, ^*; Silvio L.S. Rocha^II; Carlos A.V. Lima-Rosa^III; Guilherme F. Souza^II; Hamilton L.S. Moraes^II; Felipe O. Salle^II; Lucas B. Moraes^II; Carlos T.P. Salle^II

^ICoordenação Geral de Apoio Laboratorial, Secretaria de Defesa Agropecuária, Ministério da Agricultura, Pecuária e Abastecimento, Anexo B, Sala 430, Brasília, DF 70043-900, Brazil

^IICentro de Diagnóstico e Pesquisa em Patologia Aviária, Departamento de Medicina Animal, Faculdade de Veterinária, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves 8824, Porto Alegre, RS 91540-900, Brazil

^IIIDepartamento de Produção Animal e Alimentos, Centro de Ciências Agroveterinárias (CAV), Universidade do Estado de Santa Catarina (UDESC), Av. Luiz de Camões 2090, Bairro Conta Dinheiro, Lages, SC 88520-000, Brazil

ABSTRACT

The virulence mechanisms of avian pathogenic Escherichia coli (APEC) have been continually studied and are believed to be multi-factorial. Certain properties are primarily associated with virulent samples and have been identified in avian isolates. In this study a total of 61 E. coli, isolates from chicken flocks with respiratory symptomatology, were probed by Polimerase Chain Reation (PCR) for the presence of genes responsible for the adhesion capacity, P fimbria (papC) e F11 fimbria (felA), colicin production (cvaC), aerobactin presence (iutA), serum resistance (iss), temperature-sensitive hemagglutinin (tsh), and presence of K1 and K5 capsular antigens (kpsII). The iss gene was detected in 73,8%, tsh in 55,7%, iutA in 45,9%, felA in 39,3%, papC in 24,3%, cvaC in 23% and kpsII in18%.

Index terms:Escherichia coli, virulence, PCR, poultry, pathogenicity.

RESUMO

Os mecanismos de virulência das amostras de Escherichia coli potencialmente patogênicas para aves (APEC) têm sido continuamente estudados e acredita-se ser multifatorial. Certas propriedades são associadas primariamente a amostras virulentas e vêm sendo identificadas em amostras de E. coli isoladas de aves. Neste estudo um total de 61 amostras de E. coli, isoladas de frangos de corte com problemas respiratórios, foram testadas através da Reação em Cadeia da Polimerase (PCR), para a presença dos genes responsáveis pela capacidade de adesão, fimbria P (papC) e fimbria F11 (felA), produção de colicinas (cvaC), presença de aerobactina (iutA), resistência sérica (iss), hemaglutinina temperatura sensível (tsh) e presença de dos antígenos capsulares K1 e K5 (kpsII). O gene iss foi detectado em 73,8%, tsh em 55,7%, iutA em 45,9%, felA em 39,3%, papC em 24,3%, cvaC em 23% e kpsII em 18%.

Termos de indexação:Escherichia coli, virulência, PCR, patogenicidade, frango de corte.

INTRODUCTION

The increasing competition in meat markets gives rise to the necessity of more and more efficient and competitive production. In this scenario, production rates are fundamental to surveillance and competition of poultry proces-sing plants. These rates comprise fundamentally the sanitary aspects of the breeding stock.

Escherichia coli, which is part of the normal microbiota of birds (Bettelheim 1994) in intestinal and respiratory tracts (Morris & Sojka 1985), was forgotten as a potential pathogen. However, lesions in which E. coli is the primary and often the secondary agent cause economic damage due to lower corporal development, insufficient feed conversion, increasing mortality, higher cost with medicine, and condemnation of carcasses.

In Brazil, from 2001 to 2005, the condemnation of part of carcasses or entire carcasses because of the presence of lesions, where E. coli could be the responsible agent, caused losses estimated to 58 million dollar, 39 millions of which by systemic lesions (Brasil 2006).

Considering that 10-15% of E. coli samples can be potentially pathogenic (Barnes & Gross 1997), turned urgent and decisive for the occurrence of the disease the knowledge of interaction of factors connected with handling, nutrition, bird genetics, immunodeficiency, and especially bacterial genetics.

The virulence mechanisms of avian pathogenic E. coli (APEC) have been continually studied and are believed to be multifactorial. Certain properties are primarily associated with virulent samples and have been identified in avian isolates. The most frequently mentioned are: adhesion capacity (pap and fel), colicin production (cva), aerobactin presence (iut), serum resistance (iss) (Barnes 1997), hemagglutinin sensitive temperature (tsh), and the presence of certain capsular antigens (kps) (La Ragione & Woodward 2002).

MATERIALS AND METHODS

Bacterial samples

Sixty-one Escherichia coli isolates from 58 chicken flocks with respiratory symptomatology and post-mortem lesions compatible with colibacillosis were studied. The samples were stored in BHI (Brain Heart Infusion/ Oxoid, England) with glycerol at 30% in a freezer at -20ºC. The sample motility was verified by use of SIM Medium (Sulfuric acid indol motility/Merck, Germany).

Virulence genes

E. coli was probed by the Polimerase Chain Reaction (PCR) for the presence of genes responsible for the adhesion capacity, P fimbriae (papC) and F11 fimbriae (felA), colicin production (cvaC), aerobactin presence (iutA), serum resistance (iss), temperature-sensitive hemaglutinin (tsh) and presence of K1 and K5 capsular antigens (kpsII).

PCR

Extraction of the DNA. One milliliter of suspension from a bacterial culture in BHI of 24h at 37ºC was collected and centrifuged for 5 minutes. The supernatant was rejected and 800µl of the milliQ water was added. After having been homogenized, the samples were submitted to new centrifugation in the same conditions previously mentioned. The supernatant was rejected and 80µl of milliQ water was added. The samples were then submitted to a temperature of 96ºC for 10 minutes in water bath. The supernatant was removed and maintained frozen until the moment of analysis.

Mix. For the genes iutA and cvaC were used 11.2µl of milliQ water, 2.5µl of PCR buffer 10x (Lab Trade, Brazil), 2µl of dNTP mix with 2.5mM of each nucleotide (Invitrogen Life Technologies, USA), 2µl of 50mM MgCl2, 1µl of 20pM each primers (Invitrogen Life Technologies, USA), 0.3µl of 5U Taq DNA polymerase (Lab Trad., Brazil) and 5µl of template DNA.

For the genes felA, kpsII, papC, tsh and iss were used 11.95µl of milliQ water, 2.5µl of PCR buffer 10x (Lab Trade, Brazil), 2µl of dNTP mix with 2.5mM of each nucleotide (Invitrogen Life Technologies, USA), 1.25µl of 50mM MgCl2 (Lab Trade, Brazil), 1µl of 20pM each primers (Invitrogen Life Technologies, USA), 0.3µl of 5U Taq DNA polymerase (Lab Trade, Brazil) and 5µl of template DNA.

The primers are reported on Table 1.

Amplification. The conditions of PCR, the sequence of the primers and the size of the amplified fragment for each studied gene are described on Table 1. The tests were done in termal cycler PCT-100 (MJ Research) and the amplified DNA was visualized in agarose gel at 1.2% (Invitrogen Life Technologies, USA) contends ethidium bromide (Sigma, USA).

Reference Escherichia coli strains. BK 324 (cvaC, iss, felA, papC, tsh), IAPAR 1315 (iutA) e ATCC 35278 (kpsII).

RESULTS AND DISCUSSION

Many researchers have shown that mechanisms for patho-genicity of APECs are directly related to the interaction of several factors. Several authors dedicated themselves to these studies, and in spite of frequency variations, the following main factors are significant: adhesion capacity, colicin production, aerobactin presence, serum resistance, sensitive temperature hemagglutinin, and the presence of certain capsular antigens (Dho & Lafont 1982, Naveh et al. 1984, Rocha et al. 2002, Ngeleka et al. 2002, Brito et al. 2003, Delicato et al. 2003, McPeake et al. 2005).

In this study the presence of some genes, which are determinant in virulence and motility of litter isolates, was found. The frequency is shown on Table 2.

In colicin producing samples, the genetic determiners and the proteins that accompany them are located in plasmids, which are called Col factors (Luria 1987). Colicin V, unlike the other ones, is found mainly in virulent bacteria involved in extra-intestinal infections affecting humans and animals (Gilson et al. 1987, Lior 1994) and it inhibits the bacterial growth, interfering with the potential of membrane formation (Yang & Konsky 1984).

In this study cvaC gene and ColV plasmid structural gene were detected in 23% of the samples. Only Blanco et al. (1997) found similar results (22%), while other authors obtained higher percentage. McPeake et al. (2005) reported 99.1% and Rodriguez-Siek et al. (2005) 66.8%. In Brazilian samples the gene has been detected in 35% of them (Delicato et al. 2003).

Serum resistance is mediated by the lipopolysaccharid structural surfaces (LPS) of the capsule and other proteic membranes, the presence of outer membrane protein (Gross 1994) and K1 and K5 antigens, which are codified by kps genes (Johnson et al. 2005). Some plasmids are able to transmit serum resistance to sensitive receptive cells. Iss genes were identified in pColV-I-K94 plasmids, the protein of which is related to cytotoxic complex inhibition (Bins at al. 1979). The results of the present study are similar to the 72.8% reported by McPeake et al. (2005), the77% reported by Pfaff-McDonough et al. (2000), and the higher percentage of 81.5% found by Rodriguez-Siek et al. (2005).

The property of invading and multiplying presented by pathogens is influenced by iron availability, which is essential for growth in living cells (Neilands et al. 1985). The aerobactin system enables microorganisms to grow in iron free media at low concentration. E. coli especially uses this way of capture and transport (Rohrbach et al. 1995, Braun 2003).

The presence of operon aerobactin is in general related to ColV plasmids, although it can be chromosomal (Linggood et al. 1987, Johnson 1991). Table 2 shows that iut A gene, which codifies outer membrane protein aerobactin receptor, was detected in 45.8% of the samples. Gomis et al. (2001) reached similar results (46.8%). Rodriguez-Siek et al. (2005) and Delicato et al. (2003) got 80.2% and 63%. Vandekerchove et al. (2005) presented a lower result (23%). Rodriguez-Siek et al. (2005) detected higher results, 41.2%.

A serologic variant of P fimbriae (F11) is codified by the felA operon (De Ree et al. 1985). In samples analyzed in the present study, 39.3% were positive for the presence of this gene. Controversial results are reported by other authors, of them Delicato et al. (2003) who got 12%, while Rodriguez-Siek et al. (2005) found 78%.

Provence & Curtiss (1994) reported hemagglutination in E. coli samples which was detected only at 26-30ºC, and therefore was called sensitive temperature hemagglutinin and identified as the tsh gene. Its role in the pathogenicity of avian isolates still demands investigation, although its presence in isolated samples obtained from diseased birds has been reported by various authors. In the present work 55.7% of the samples analyzed from litter were positive. Ewers et al. (2004) found 53.3%, while other authors reported 85.3% (Janben et al. 2001), 93.95% (McPeake 2005), and 99% (Ngeleka et al. 2002).

The flagella, which are thin surface appendixes, give motility to Gram positive and negative bacteria in aqueous media. Their rotating movements allow microorganisms to approach adjacent epithelial cells, crossing the mucus barrier and causing adhesion, multiplication, colonization and infection (La Ragione & Woodward 2002). In the present study motility was detected in 54.1% of the samples, higher than 36.8% reported by McPeake et al. (2005).

The diversity of genes associated with pathogenicity, which was found in the present study and in other scientific articles, suggests the existence of a real interaction among APEC virulence factors. However, despite the technological progress, the contribution to their patho-genicity has not been established so far. Consequently, no significant advance and objective answers to profes-sionals have been obtained.

The growing limitations for the use of antimicrobics in avian production, do not allow professionals to be informed about the E. coli pathogenic potential, to direct their procedures. For this to happen, new technology is necessary to detect the pathogenicity of isolates in an objective, fast and efficient way.

Acknowledgements.- To Dr. Benito Guimarães de Brito (Instituto de Pesquisas Veterinárias Desiderio Finamor/IPVDF) for supplying the Escherichia coli strains BK 324 e IAPAR 1315, and to Dr. Marisa Ribeiro de Itapema Cardoso (Faculdade de Veterinária-Universidade Federal do Rio Grande do Sul/UFRGS) for supplying the E. coli strains ATCC 35278. To Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) for the support through a scholarship.

Received on July 20, 2006.

Accepted for publication on January 15, 2008.

References

• Barnes H.J. 1994. Pathogenesis of respiratory Escherichia coli and Pasteurella multocida infections in poultry. Respiratory Diseases of Chicken and Turkeys. AAAP, San Francisco, California, USA.
• Barnes h.j. & Gross W.B. 1997. Colibacillosis, p.131-141. In: Calnek, B.W. (ed.), Diseases of Poultry. University Press, Ames, Iowa, USA.
• Bettelheim K.A. 1994. Biochemical characteristics of Escherichia coli, p.3-30. In: Gyles C.L. (ed.), Escherichia coli in Domestic animals and humans. CAB International, Wallington, Oxon, UK.
• Binns M.M., Davies D.L. & Hardy K.G. 1979. Cloned fragments of the plasmid ColV, I-K94 specifying virulence and serum resistance. Nature 279:778-781.
• Blanco J.E., Blanco M., Mora A. & Blanco J. 1997. Production of toxins (enterotoxins, verotoxins, and necrotoxins) and colicins by Escherichia coli strains isolated from septicemic and healthy chickens: relationship with in vivo pathogenicity. J. Clin. Microbiol. 35:2953-2957.
• Brasil 2006. Setor de Estatística, Serviço de Inspeção Federal, Delegacia Federal de Agricultura e Reforma Agrária, Ministério da Agricultura, do Abastecimento e da Reforma Agrária, Brasília, DF.
• Braun V. 2003. Iron uptake by Escherichia coli Front Biosci. 8:1409-1421.
• Rocha A.C., Silva A.B., de Brito A.B., Moraes H.L., Pontes A.P., Ce M.C., Nascimento V. & Salle C.T. 2002. Virulence factors of avian pathogenic Escherichia coli isolated from broilers from the south of Brazil. Avian Dis. 46:749-753.
• Brito B.G., Gaziri L.C. & Vidotto M.C. 2003. Virulence factors and clonal relationships among Escherichia coli strains isolated from broiler chickens with cellulitis. Infect. Immun. 71:4175-4177.
• De Ree J.M., Schwillens P. & Van den Bosch J.F. 1985. Monoclonal antibodies that recognize the P fimbriae F71, F72, F9, and F11 from uropathogenic Escherichia coli. Infect. Immun. 50:900-904.
• Delicato E.R., Brito B.G., Gaziri L.C. & Vidotto M.C. 2003. Virulence-associated genes in Escherichia coli isolates from poultry with colibacillosis. Vet. Microbiol. 94:97-103.
• Dho M., Lafont J.P. 1982. Escherichia coli colonization of the trachea in poultry: comparison of virulent and avirulent strains in gnotoxenic chickens. Avian Dis. 26:787-797.
• Ewers C., Janssen T., Kiessling S., Philipp H.C. & Wieler L.H. 2004. Molecular epidemiology of avian pathogenic Escherichia coli (APEC) isolated from colisepticemia in poultry. Vet. Microbiol. 104:91-101.
• Gilson L., Mahanty H.K. & Kolter R. 1987. Four plasmid genes are required for colicin V synthesis, export, and immunity. J. Bacteriol. 169:2466-2470.
• Gomis S.M., Riddell C., Potter A.A. & Allan B.J. 2001. Phenotypic and genotypic characterization of virulence factors of Escherichia coli isolated from broiler chickens with simultaneous occurrence of cellulitis and other colibacillosis lesions. Can. J. Vet. Res.65:1-6.
• Gross W.G. 1994. Diseases due to Escherichia coli in Poultry, p.237-260. In: Gyles C.L. (ed.), Escherichia coli in Domestic Animals and Humans. CAB International, Wallington, Oxon, UK.
• Gyles C.L. 1994. Escherichia coli in Domestic animals and humans. CAB International, Wallington, Oxon, UK.
• Janben T., Schwarz C., Preikschat P., Voss M., Philipp H.C. & Wieler L.H. 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.
• Johnson J.R. 1991. Virulence factors in Escherichia coli urinary tract infection. Clin. Microbiol. Rev. 4:80-128.
• Johnson J.R., Kuskowski M.A., Gajewski A., Soto S., Horcajada J.P., Jimenez de Anta M.T. & Vila J. 2005. Extended virulence genotypes and phylogenetic background of Escherichia coli isolates from patients with cystitis, pyelonephritis, or prostatitis. J. Infect. Dis. 191:46-50.
• La Ragione R.M. & Woodward M.J. 2002. Virulence factors of Escherichia coli serotypes associated with avian colisepticaemia. Res. Vet. Sci. 73:27-35.
• Linggood M.A., Roberts M., Ford S., Parry S.H. & Williams P.H. 1987. Incidence of the aerobactin iron uptake system among Escherichia coli isolates from infections of farm animals. J. Gen. Microbiol. 133:835-842.
• Lior H. 1994. Classification of Escherichia coli, p.31-72. In: Gyles C.L. (ed.), Escherichia coli in Domestic Animals and Humans. CAB Inter-national, Wallington, Oxon, UK.
• Luria S.E. & Suit J.L. 1987. Colicins and COL plasmids, p.1615-1624. In: Neidhardt F.C. (ed.), Escherichia coli and Salmonella Typhimurium: Cellular and molecular biology. Vol.2. American Society of Micro-biology, Washington, DC.
• McPeake S.J., Smyth J.A. & Ball H.J. 2005. Characterization of avian pathogenic Escherichia coli (APEC) associated with colisepticaemia compared to faecal isolates from healthy birds. Vet. Microbiol. 110:245-253.
• Morris J.A. & Sojka W.J. 1985. The virulence of Escherichia coli, p.47-77. In: Sussman M. (ed.), Escherichia coli as a Pathogen in Animals. Academic Press, London.
• Naveh M.W., Zusman T., Skutelsky E. & Ron E.Z. 1984. Adherence pili in avian strains of Escherichia coli: effect on pathogenicity. Avian Dis. 28:651-661.
• Neilands J.B., Bindereif A. & Montgomerie J.Z. 1985. Genetic basis of iron assimilation in pathogenic Escherichia coli Curr. Top. Microbiol. Immunol. 118:179-195.
• Ngeleka M., Brereton L., Brown G. & Fairbrother J.M. 2002. Pathotypes of avian Escherichia coli as related to tsh-, pap-, pil-, and iuc-DNA sequences, and antibiotic sensitivity of isolates from internal tissues and the cloacae of broilers. Avian Dis. 46:143-152.
• Pfaff-McDonough S.J., Horne S.M., Giddings C.W., Ebert J.O., Doetkott C., Smith M.H. & Nolan L.K. 2000. Complement resistance-related traits among Escherichia coli isolates from apparently healthy birds and birds with colibacillosis. Avian Dis. 44:23-33.
• Provence D.L. & Curtiss R. 1994. Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain. III. Infect. Immun. 62:1369-1380.
• Rodriguez-Siek K.E., Giddings C.W., Doetkott C., Johnson T.J., Fakhr M.K. & Nolan L.K. 2005. Comparison of Escherichia coli isolates implicated in human urinary tract infection and avian colibacillosis. Microbiology. 151:2097-2110.
• Rohrbach M.R., Braun V. & Koster W. 1995. Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB. J. Bacteriol. 177:7186-7193, 1995
• Vandekerchove D., Vandemaele F., Adriaensen C., Zaleska M., Hernalsteens J.P., De Baets L., Butaye P., Van Immerseel F., Wattiau P., Laevens H., Mast J., Goddeeris B. & Pasmans F. 2005. Virulence-associated traits in avian Escherichia coli: comparison between isolates from colibacillosis-affected and clinically healthy layer flocks. Vet. Microbiol. 108:75-87.
• Yang C.C. & Konisky J. 1984. Colicin V treated Escherichia coli does not generate membrane potential. J. Bacteriol. 158:757-759.

Publication Dates

• Publication in this collection
  13 June 2008
• Date of issue
  Mar 2008

History

• Accepted
  15 Jan 2008
• Received
  20 July 2006