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Brazilian Journal of Microbiology

Print version ISSN 1517-8382On-line version ISSN 1678-4405

Braz. J. Microbiol. vol.37 no.3 São Paulo July/Sept. 2006

http://dx.doi.org/10.1590/S1517-83822006000300033 

VETERINARY MICROBIOLOGY

 

Some adhesins of avian pathogenic Escherichia coli (APEC) isolated from septicemic poultry in Brazil

 

Algumas adesinas de Escherichia coli aviária (APEC) isoladas de aves com colisepticemia no Brasil

 

 

Terezinha KnöblI,II; Tânia Aparecida Tardelli GomesIII; Mônica Aparecida Midolli VieiraIII; Fernando FerreiraII; José Américo BottinoII,; Antônio José Piantino FerreiraII,*

IFaculdade de Medicina Veterinária, Faculdades Metropolitanas Unidas, São Paulo, SP, Brasil
IIFaculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP, Brasil
IIIDepartamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, SP, Brasil

 

 


ABSTRACT

Three hundred and fifty strains of E. coli isolated from septicemic poultry from seven states of Brazil were examined for presence of nine adhesion-encoding genes, hemagglutination and adherence to chicken tracheal cells (in vitro). Analysis of the strains by colony hybridization tests demonstrated that 93.7% of the isolates were fim +, 17% pap+ and 5.7% were sfa+. The mannose sensitive fimbriae occur with similar frequency in APEC isolated from all Brazilians states, while significant differences among pap and sfa genes distributions were observed. The results showed that 0.85% and 0.28% of APEC were positive for genes that encoded enteroaggregative adhesins and EPEC adherence factor, respectively. None of APEC was positive for DA, afa, Bfp and Eae probes. The adherence to chicken tracheal cells showed 96% positive strains, while hemagglutination assays showed 26.5% of the isolates were mannose sensitive and 21.7% were mannose resistant.

Key words: APEC, Escherichia coli, adhesins, colibacillosis, poultry


RESUMO

Trezentas e cinqüenta amostras de E. coli isoladas de aves com septicemia em sete estados do Brasil foram examinadas para a presença de nove genes codificadores de adesinas, hemaglutinação e aderência em células da traquéia (in vitro). A análise das amostras pela hibridização de colônias demonstrou que 93,7% dos isolados eram fim +, 17% pap+ e 5,7% eram sfa+. As fímbrias manose sensíveis apresentaram uma distribuição uniforme em todos os estados do Brasil. No entanto, diferenças significativas na distribuição dos genes pap e sfa foram observadas. Os resultados mostraram que 0,85% e 0,28% das APEC foram positivas para os genes que codificam as adesinas enteroagregativas e o fator de aderência de EPEC, respectivamente. Nenhuma amostra foi positiva para as sondas DA, afa, Bfp e Eae. A aderência em células de traquéia de aves revelou 96% de amostras positivas, enquanto os testes de hemaglutinação mostraram 26,5% dos isolados mannose sensíveis e 21,7% manose resistentes.

Palavras-chave: APEC, Escherichia coli, adesinas, colibacilose, ave


 

 

INTRODUCTION

Avian Pathogenic Escherichia coli (APEC) are associated with extra intestinal infections and development of septicemia in broilers. Colibacilosis is an opportunistic disease, responsible for severe economic losses for the poultry industry due to a lowered production, increased mortality rate, carcass condemnation and cost of treatment (3,5).

Extra intestinal E. coli strains encode many adhesins that promote the attachment of the bacteria to cell receptors. These virulence factors are very important for the host infection and to development of septicemia (10,24,29,31-34,39,40). Type 1 fimbriae have been involved with the initial stages of the upper respiratory colonization, whereas the P fimbriae are involved in colonization of the internal organs (31).

Type 1 fimbriae are found in many different species of Enterobacteriaceae and are characterized by their ability to mediate agglutination of guinea pig erythrocytes in the absence of a-D-mannose, but not in its presence. The role of type 1 fimbriae in colibacillosis has been associated with mucus adherence and trachea and intestinal tract colonization (8,11,13,28,31,35).

P fimbriae are mannose resistant hemagglutinating fimbriae present in E. coli strains causing urinary tract infections in humans and also may be expressed by some E. coli of avian origin. They are associated with internal organs colonization, septicemia and lethality in one-day-old chicks (12,31).

Epidemiological studies with APEC have shown the presence of other selected genes for fimbrial and afimbrial adhesins of human origin. The role of these adhesins in pathogenesis of colibacillosis has not been elucidated, but poultry may act as a reservoir for human pathogenic E. coli (12,19,20,24,30,36,40).

The purpose of this survey was to investigate the distribution of adhesion-encoding genes among avian pathogenic E. coli (APEC) isolated from septicaemic poultry in seven Brazilians states.

 

MATERIALS AND METHODS

Bacterial strains and growth conditions

A total of 350 E. coli strains were isolated from different poultry farms between 1994 and 2004 in seven states of Brazil - Ceará, Pernambuco, Minas Gerais, São Paulo, Paraná, Santa Catarina and Rio Grande do Sul.

The strains were isolated from the liver of broilers with colisepticemia. Standard bacteriological methods were employed for isolation and identification of the organism (3). All strains were stored at -80ºC in Brain Heart Infusion (BHI) broth (Difco Laboratories, Detroit, MI, USA) containing 15% glycerol, added after incubation.

Colony Hybridization

The colony hybridization assays were performed as described by Maas (27). The test strains were examined using specific DNA probes labeled with [a -32P]-dATP by Nick translation. The DNA probe donors, recombinant plasmids, fragments size and relevant literature are given in Table 1. Detection of pap, afa, and sfa genes involved oligonucleotide fragments obtained by polymerase chain reaction (Table 2). Positive and negative controls were included in all hybridization assays.

Hemagglutination tests

The presence of fimbriae was detected by the ability of strains to agglutinate erythrocytes from guinea pig, human, chicken, cattle and sheep in the presence or absence of 2% D-Mannose (14).

Tracheal ring cell preparation and adherence

To assess E. coli isolates for adherence, the bacteria were grown on colonization factor antigen agar (CFA) at 37ºC for 18-24 h and then suspended in 50 mM PBS. Ten-day-old Specific-Pathogen-Free chicks were killed humanely and the tracheas removed aseptically. Tracheal sections were cut in 4 mm length, rinsed three times in Krebs Ringer Tris saline buffer with 0.05 M Tris-HCl (pH 7.4) (18).

Adherence studies were performed in 96-well round-bottom microtiter plates. Each well received three tracheas and MEM without calf serum. Bacterial strains were incubated with tracheal rings at 37ºC for 30 min, after which they were washed with 50 mM PBS (pH 7.4) and incubated for an additional 4 h. Then, the tracheal rings were fixed with buffered formalin, processed and stained with Giemsa for examination by light microscopy. E. coli K 12 strain C600 was used as a negative control (18).

Statistical analysis

All data were analyzed by means of the software EpiInfo - Centers for Disease Control and Prevention, Atlanta, GA, USA (6). Fisher´s exact test and the c2 test were used for univariate analysis of the significance associations. Differences were considered statistically significant if P d<0.05.

 

RESULTS

The results of the DNA hybridization tests, hemagglutination assays and tracheal adherence tests are summarized in Table 3.

Type 1 probe showed that the relevant sequence was present in 328 (93.7%) isolates. These fimbriae occurred with similar frequency in strains from the seven Brazilians states.

Sixty one (17.4%) isolates were pap+ and twenty (5.7%) were sfa+. Significant differences were observed in the distribution of mannose-resistant fimbriae among several Brazilians states. The pap+ gene frequency varied from 18 and 26% in the majority of the states, while Ceará and Minas Gerais presented only 6 and 10% of pap+ APEC, respectively. The sfa genes were detected with a higher frequency in isolates from Paraná (30%) than in isolates from São Paulo (4%), Minas Gerais (4%) and Rio Grande do Sul (2%), and were absent from strains isolated from other states.

None of E. coli isolates carried the genes encoding for afimbrial adhesin (afa), diffuse adhesion (DA) and attaching and effacing lesion (eae).

The phenotypic assays showed that 93 (26.5%) strains presented mannose sensitive hemagglutination and 76 (21.7%) mannose resistant hemagglutination (Table 3). HAMS profiles varied among 12% and 46% in Brazilian states and were considered very lower than results obtained in colony hybridization for type 1 fimbriae (88% to 98%). Results of HAMR varied greatly among different Brazilian states (Table 3) and didn't correlate with results of P and S DNA positive probe.

In relation to adherence tests, 336 (96%) strains adhered on tracheal cells in absence of D-mannose. There were no significant differences among the results obtained in several Brazilian states (p<0.05).

 

DISCUSSION

Epidemiological and pathogenesis researches on the E. coli are concentrated on adhesion investigations, because fimbriae are good candidates for vaccine against APEC (23,36-38). The present study shows that the presence of mannose-resistant adhesins varies among strains isolated from different Brazilian states, while the mannose sensitive adhesins presents a uniformly distribution (Table 3).

This study also confirms previous observations that type 1 fimbriae are frequently detected in APEC (7,20,29,40). A total of 93.7% E. coli isolates were found to hybridize with fim DNA probe, but only 26.5% exhibited mannose-sensitive hemagglutination pattern (MSHA). The results of MSHA were lower than colony hybridization for type 1 fimbriae in the seven Brazilian states, suggesting that hemmaglutination assay was a low sensitive phenotypic test.

Dho and Lafont (9) have associated the virulence of avian E.coli with its ability to adhere to tracheal epithelial cells. In this study, 96% of E. coli isolates presented adherence to tracheal epithelium in absence of D-mannose, and there were no significant differences among the results obtained in several Brazilian states (p<0.05), as show the Table 3. The presence of type 1 fimbriae was correlated with tracheal cells adherence, and this phenotypic expression model was more sensitive than hemagglutination assay. However, some isolates that were positive to tracheal cells adherence didn't hybridize with fim operon, suggesting a low specificity of this adherence model.

Vandemaele et al. (37) analyzed the sequence of fimH and fimA genes in 24 isolates of APEC and demonstrated that fimH is a conserved adhesin, while fimA presents a variable sequence, although, the immunization with the binding domain of fimH does not protect chickens against avian pathogenic E. coli (38).

Pourbakhsh et al. (31) demonstrated the involvement of type 1 fimbriae in the colonization of the upper respiratory tract in experimentally inoculated chickens and suggested that P fimbriae may be involved in the colonization of internal organs and in the development of septicaemia. However, the major part of E.coli isolated from colibacillosis was negative for fimbriae P, and the mannose resistant adherence cannot always be attributed to P pili (33,40).

Epidemiological studies about APEC present relative diversity among pap frequency (7,20,24,29,33,40). For instance, the pap gene was detected in 30% of isolateds by Janben et al. (20), 23.9% of isolates by Stouder et al. (34) and 16% of isolates by Knöbl et al. (24).

In Brazil, the epidemiological studies on APEC were concentrated in the Paraná state, with 14% of E. coli pap+ isolated by Vidotto et al. (40); 18.5% by Delicato et al. (7); and 17.4% in this study. Amabile de Campos et al. (1) studied 45 APEC obtained from chickens suffering from septicemia, swollen head syndrome and omphalitis, isolated from individuals in different regions of Brazil and detected 11 (22.4%) pap positive strains. Our results suggest that the frequency of pap gene in Brazil can vary between 6 and 26%. The regional variation in the frequency of P fimbriae may be considered one limitation for protection against colibacilosis by fimbriae vaccine. Other limitation of the P pilus vaccine is the highly polymorphic nature of Pap A main subunit. Vandemaele et al. (36) showed that the papGII and papGIII sequences of APEC have high homology with human papG sequences. Moreover to mention the zoonotic consequences, the authors suggested that the conserved character makes it a promising vaccine candidate against APEC.

Stoudeur et al. (34) analyzed a collection of 1601 of extra intestinal or intestinal Escherichia coli isolated from chickens, turkeys and ducks, in Belgium, France and Spain and observed that 4.2% of strains were S-positive. Knöbl et al. (24) showed that the frequency of these fimbriae in Brazil can vary between 4 and 16% among isolates from respiratory disease and omphalitis, respectively. Chicks may become infected due to poor hygiene during handling of eggs in the hatchery. The fecal-oral route can be responsible for the widespread of infection.

Significant differences were appointed in the frequency of sfa gene in this investigation. The sfa gene was detected in 4% of isolates from São Paulo and Minas Gerais states, while in Paraná state, 30% of APEC were sfa+. Vidotto et al. (39) examined APEC isolates from Paraná state using colony hybridization and found that 40% were positive for sfaDE and 30% for facA genes. Amabile de Campos et al. (1) found sfa adhesion sequence in 4.16% of septicaemic E. coli isolated from chickens in Brazil. The epidemiology of Escherichia coli sfa+ isolated from poultry in Brazil needs to be better characterized.

The S fimbriae are able to promote the adherence of E. coli to endothelial and epithelial cells in human coroid plexus and cerebral ventriculus. The presence of S fimbriae was rarely detected in APEC and the role of these fimbriae on pathogenesis of colibacillosis had not been elucidated (19,24,34).

Certain adhesins associated with E. coli causing intestinal disease in humans are found sporadically in APEC (20,25). To our knowledge, this work is the first report of enteroaggregative E. coli isolated from poultry. The importance of these strains for poultry is unclear, but domestic animals may constitute reservoirs of strains that are pathogenic for humans. Janben et al. (20), using PCR for identifying virulence-associated genes in 150 APEC, found enteroaggregative heat stable toxin gene (astA) in 17.3% of the field strains.

EAF gene sequences in strains of avian origin are also rare, but other virulence determinants of EPEC, like eae, gene have been described by many authors (16,25,30). In this study only one strain presented the EAF sequences, but this APEC was negative for EAE probe.

The afa sequences were not found in this investigation and this result is in accordance with results obtained by Delicato et al. (7). However, the afa sequence was detected in 4.8% of isolates from avian origin by Stordeur et al. (34) and 12.5% by Amabile de Campos et al. (1).

In conclusion, the results of this study confirm the regional differences of frequency of mannose resistant adhesion genes in APEC isolated from Brazil. A more complete understanding about these fimbriae is necessary to support a vaccine programs.

 

ACKNOWLEDGMENTS

We are grateful to FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for financial support - Grant 97/3250-3.

 

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Submitted: July 28, 2005; Returned to authors for corrections: November 18, 2005; Approved: May 19, 2006

 

 

In memorian
* Corresponding author. Mailing address: Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, Av. Prof. Dr. Orlando M. Paiva, 87, Cidade Universitária. 05508-900, São Paulo, SP, Brasil E-mail: af.piantino@fmvz.usp.br

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