Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC)

Lilian Aparecida Sanches Marcelo da Silva Gomes Rodrigo Hidalgo Friciello Teixeira Marcos Paulo Vieira Cunha Maria Gabriela Xavier de Oliveira Mônica Aparecida Midolli Vieira Tânia Aparecida Tardelli Gomes Terezinha Knobl About the authors

ABSTRACT

Psittacine birds have been identified as reservoirs of diarrheagenic Escherichia coli, a subset of pathogens associated with mortality of children in tropical countries. The role of other orders of birds as source of infection is unclear. The aim of this study was to perform the molecular diagnosis of infection with diarrheagenic E. coli in 10 different orders of captive wild birds in the state of São Paulo, Brazil. Fecal samples were analyzed from 516 birds belonging to 10 orders: Accipitriformes, Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Pelecaniformes, Piciformes, Psittaciformes and Strigiformes. After isolation, 401 E. coli strains were subjected to multiplex PCR system with amplification of genes eae and bfp (EPEC), stx1 and stx2 for STEC. The results of these tests revealed 23/401 (5.74%) positive strains for eae gene, 16/401 positive strains for the bfp gene (3.99%) and 3/401 positive for stx2 gene (0.75%) distributed among the orders of Psittaciformes, Strigiformes and Columbiformes. None of strains were positive for stx1 gene. These data reveal the infection by STEC, typical and atypical EPEC in captive birds. The frequency of these pathotypes is low and restricted to few orders, but the data suggest the potential public health risk that these birds represent as reservoirs of diarrheagenic E. coli.

Keywords:
Diarrheagenic E. coli; Atypical EPEC; STEC; Avian diseases

Introduction

Many zoonoses are associated with ecological imbalances as a result of deforestation, human population expansion, changes in agricultural practices and encroachment on wildlife habitats.11 Chomel BB, Belotto A, Meslin FX. Wildlife exotic pets, and emerging zoonoses. Emerg Infect Dis. 2007;13:1-8. Urban areas represent impacted environment and wild animals are maintained in captivity, living in zoos. Captive wildlife animals are very susceptible to opportunistic diseases and they may act as reservoir of pathogenic bacteria.22 Ahmed AM, Motoi Y, Sato M, et al. Zoo animals as reservoir of Gram-negative Bacteria harboring integrons and antimicrobial resistance genes. Applied Envirom Microbiol. 2007;:.
https://doi.org/10.1128/AEM.01054-07...
,33 Davies YM, Cunha MPV, Oliveira MGX, et al. Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerines and psittacine birds. Avian Pathol. 2016;45:194-201.

Escherichia coli can be considered the most prevalent opportunistic enterobacteria in captive animals and were associated with systemic disease in birds.44 Mattes BR, Consiglio SAS, Almeida BZ, et al. Influência da Biossegurança na colonização intestinal por Escherichia coli em psitacídeos. Arq Inst Biol. 2005;72:13-16. Airsaculitis and sepsis are often caused by avian pathogenic Escherichia coli (APEC), which are considered as Extraintestinal pathogenic E. coli (ExPEC) pathotype.55 Cunha MPV, Oliveira MGX, Oliveira MC, et al. Virulence profiles, phylogenetic background and antibiotic resistance of Escherichia coli isolated from turkeys with airsacculitis. Sci World J. 2014;2014:1-8. The pathogenesis of enteritis by E. coli in birds is still unclear, but the presence of diarrheagenic strains may represent a public health risk. Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) represent two of at least six pathotypes of human diarrheagenic E. coli that affect birds and may be considered zoonotic pathogens.66 Farooq S, Hussain MA, Bhat MA, Wani SA. Isolation of atypical enteropathogenic Escherichia coli and Shiga toxin 1 and 2f-producing Escherichia coli from avian species in India. Lett Appl Microbiol. 2009;48:692-697.,77 Gioia Di-Chiacchio RM, Cunha MPV, Sturn RM, et al. Shiga toxin-producing Escherichia coli (STEC): zoonotic risks associated with psittacine pet birds in home environment. Vet Microbiol. 2016;184:27-30.

The EPEC pathotype leads to a high child mortality rate in developing countries. Diarrhea is a consequence of loss of intestinal microvillus, after bacterial adherence on enterocytes, with ensuing attaching and effacing (A/E) lesions.88 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26:822-880. The AE lesion depends on intimin, an outer membrane protein, codified by the eae gene, which is present in the pathogenicity island termed the locus of enterocyte effacement (LEE).99 Vieira MAM. Ilhas de Patogenicidade. O Mundo da Saúde. 2009;33:406-414. The identification of typical EPEC strains (tEPEC) is performed by molecular detection of the eae and bfp genes ("bundle-forming pili" encoded by the EAF plasmid). The EPEC are regarded as atypical (aEPEC) in case of absence of the EAF plasmid, making it eae+ and bfp-.1010 Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis. 2002;8:508-513.

The attaching and effacing genes on the LEE may be also present in STEC, but Shiga toxin production is considered the most relevant virulence factor of this diarrheagenic E.coli patothype.88 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26:822-880.,1111 Persad AK, Lejeune JT. Animal reservoirs of Shiga toxin-producing Escherichia coli. Microbiol Spect. 2014;2:1-14. Shiga toxins can be classified into two types, Stx1 (subtypes a, c and d) and Stx2 (subtypes a to g). Both toxins are encoded on prophages that are integrated into the chromosome and lead to inhibition of protein synthesis, causing cell death.88 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26:822-880.

In humans, STEC infection causes hemorrhagic colitis following injury of the intestinal epithelium, induced by Shiga-toxin production. Stx-2 toxin is more toxic than Stx-1, and is often associated with hemolytic uremic syndrome.88 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26:822-880. The transmission routes of STEC include ingestion of contaminated food or water and contact with infected companion animals (dogs, cats and birds).77 Gioia Di-Chiacchio RM, Cunha MPV, Sturn RM, et al. Shiga toxin-producing Escherichia coli (STEC): zoonotic risks associated with psittacine pet birds in home environment. Vet Microbiol. 2016;184:27-30.,88 Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev. 2013;26:822-880.,1111 Persad AK, Lejeune JT. Animal reservoirs of Shiga toxin-producing Escherichia coli. Microbiol Spect. 2014;2:1-14.

The aim of this study was to search for the presence of EPEC and STEC isolates in captive birds from different orders located at zoos from São Paulo, state, Brazil.

Material and methods

Birds

This project was approved by the Ethics Committee of São Paulo University (2984230514) and authorized for scientific purposes (SISBIO 43541-1). We examined a total of 516 fecal samples isolated from captive birds belonging to 10 orders (including 70 species): Accipitriformes (hawk, n = 14), Galliformes (guan and curassow, n = 50), Anseriformes (duck and goose, n = 80), Psittaciformes (macaw, parrot and parakeet, n = 99), Passeriformes (canary and thrush, n = 88), Falconiformes (falcon, n = 46), Strigiformes (owl, n = 48), Columbiformes (pigeons, n = 72); Piciformes (toucan and aracari, n = 10) Pelecaniformes (pelican and egret, n = 9).

The samples were collected from September 2013 to June 2015, in two municipal zoos located in São Paulo State, Brazil. Fecal swabs were seeded in Amies transport media and sent to the laboratory, under refrigerated conditions.

Culture and Identification of E. coli

The fecal samples were enriched in brain heart infusion broth, seeded on MacConkey agar, and incubated at 37 °C for 24 h. Bacteria were identified by biochemical tests, using an Enterokit (Probac® - São Paulo, Brazil).

PCR Amplification for virulence genes

Search for virulence genes in the diarrheagenic E. coli isolates performed by PCR for amplification of eae (454 bp), bfp (550 bp), stx1 (349 bp) and stx2 (110 bp) genes, according to the method described by Costa et al. (2010).1212 Costa ARF, Lima KVB, Sousa CO, Loureiro ECB. Desenvolvimento de PCR multiplex para a detecção e diferenciação de categorias de Escherichia coli diarreiogênicas. Rev Pan-Amaz Saúde. 2010;1:77-84. The following strains were used as control of the PCR: E. coli DH5α (negative control); O157:H7 (STEC positive control) and O55:H7 (EPEC positive control).

The DNA extraction was performed as described by Boom et al. (1990).1313 Boom R, Sol CJA, Salimans MMM. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28:453-459. The amplification mixture consisted of Tris-HCl buffer (pH 8.3) 10 mM, MgCl2, deoxynucleotide triphosphates 200 mM, pairs of primers, Taq DNA polymerase 0.5 U, and ultrapure water autoclaved in a final volume of 25 µl. Amplified products were separated in 1.5% agarose gel and examined after stained with BlueGreen® (LGC Biotecnologia, São Paulo, Brazil). A 100 bp DNA ladder (LGC Biotecnologia, São Paulo, Brazil) was used as a molecular size marker.

Results

A total of 401 isolates were identified as E. coli. After PCR investigation, 23/401 isolates were positive for eae, 16/401 positive for bfp and 3/401 positive for stx2 genes (Table 1). None of strains (0/401) were positive for stx1 gene.

Table 1
Isolation of E. coli and virulence genes distributed according to the orders of captive wild birds. São Paulo, 2013-2015.

The pathotype classification of the isolates is presented in Table 2. These results showed that atypical EPEC were detected in 3/10 orders, including Psittaciformes (2/99 birds), Columbiformes (2/72 birds) and Strigiformes (1/48 birds). Typical EPEC were detected in 2/10 orders, including Psittaciformes (11/99 birds) and Columbiformes (4/72 birds). STEC was detected only in Columbiformes, present in 3/72 birds.

Table 2
Pathotypes of diarrheagenic E. coli distributed according to the orders of captive wild birds. São Paulo, 2013-2015.

The total prevalence of diarrheagenic E. coli strains was 23/401 (5.74%), which included 13/76 (17.03%) from Psittaciformes, 9/72 (12.48%) from Columbiformes and 1/37 (2.7%) from Strigiformes.

Discussion

Brazil has great wildlife biodiversity. A wide range of birds species are also kept in zoos for entertainment, education, search and conservation. Our study analyzed 10 orders with 70 species of birds for the presence of diarrheagenic E. coli. Unfortunately, due to the small number of birds in some orders, a fair sampling was somewhat compromised. Our results demonstrate that molecular techniques were useful for diagnosis of the diarrheagenic E. coli pathotypes, identifying captive birds infected by typical or atypical EPEC and STEC.

The results showed that EPEC were found in 3/10 orders, including Psittaciformes (2/99 birds), Columbiformes (2/72 birds) and Strigiformes (1/48 birds). Likewise, Kobayashi et al. (2009) evaluated the prevalence of eae- and stx-positive E. coli strains in 447 wild birds belonging to 62 species in Tokyo.1414 Kobayashi H, Kanazaki M, Hata E, Kubo M. Prevalence and characteristics of eae-and-stx-positive strains of Escherichia coli from wild birds in the Immediate Enviroment of Tokyo Bay. Appl Environ Microbiol. 2009;75:292-295. Eae-positive strains were found in 7/10 orders: Columbiformes, Passeriformes, Anseriformes, Ciconiformes, Procellariformes, Pelecaniformes and Galliformes. However, the prevalence of eae-positive strains in Tokyo was 25% (11/447), higher than the prevalence of this study (5.74%). Psittaciformes were not included in a Tokyo survey, but Passeriformes, Columbiformes and Pelecaniformes were implicated as a reservoir of EPEC.1414 Kobayashi H, Kanazaki M, Hata E, Kubo M. Prevalence and characteristics of eae-and-stx-positive strains of Escherichia coli from wild birds in the Immediate Enviroment of Tokyo Bay. Appl Environ Microbiol. 2009;75:292-295. In Brazil, none of the Passeriformes, Pelecaniformes or Anseriformes investigated were infected by EPEC. However, we found one atypical EPEC in Strigiformes (1/37 owl). To our knowledge, this is the first report of infection by EPEC in owls.

Relative analyses showed that Psittaciformes is the most prevalent order of birds positively infected by EPEC (Table 2), with 14.4% of typical EPEC and 2.63% of atypical EPEC. The EPEC infection of psittacine birds in Brazil was reported previously in parrots (Amazona aestiva, Amazona amazonica) and macaws (Anodorhynchus leari, Guarouba guarouba) with prevalence ranging from 2.27% in free-ranging birds1515 Saidenberg ABS, Guedes NMR, Seixas GHF, et al. A survey for Escherichia coli virulence factors in asymptomatic free-ranging parrots. Int Schol Res Network. 2012;2012:1-6. to 6.5% for captive psittacine birds.1616 Saidenberg ABS, Teixeira RHF, Guedes NMR, Allgayer Melville PA, Benites NR. Molecular detection of enteropathogenic Escherichia coli in asyntomatic captive psittacines. Pesq Vet Bras. 2012;32:922-926.

Marietto-Gonçalves et al. (2011) investigated swabs from 86 psittacidaes recovered from illegal wildlife trade in Brazil and found only one strain (1/86 - 1.1%) classified as typical EPEC, isolated from a blue-fronted parrot.1717 Marietto-Gonçalves G, Almeida SM, Rodrigues J. Presence of a human diarrheagenic Escherichia coli clone in captivity kept Psittacidaes. Open Microbiol. 2011;5:72-75. We believe that the high prevalence reported in our study (11.11%) is related to zoo enclosures that allow intense contact between birds, mammals and park visitors. Bacterial diversity was reported previously to be significantly lower in wild parrots and the composition of cloacal bacterial microbiota might undergo significant changes in captive birds if they are overexposed to contact with mammals.1818 Xenoulis PG, Gray PL, Brightsmith D, et al. Molecular characterization of the cloacal microbiota of wild and captive parrots. Vet Microbiol. 2010;146:320-325.

Farooq et al. (2009) found a high prevalence of atypical EPEC (15.56%) from avian species in India.66 Farooq S, Hussain MA, Bhat MA, Wani SA. Isolation of atypical enteropathogenic Escherichia coli and Shiga toxin 1 and 2f-producing Escherichia coli from avian species in India. Lett Appl Microbiol. 2009;48:692-697. The frequency was greater in farmed animals (chicken and duck - 27/112) than in pigeons (6/100), but the authors still believe that pigeons act as an infectious source for commercial poultry. Our survey also highlights the role of Columbifomes as a reservoir of EPEC and STEC. We detected 6/72 EPEC and 3/72 STEC strains in feral pigeons (Columba livia). This data are similar to those reported by Kobayashi et al. (2009), with 5/67 EPEC and 2/67 STEC strains in Tokyo.1414 Kobayashi H, Kanazaki M, Hata E, Kubo M. Prevalence and characteristics of eae-and-stx-positive strains of Escherichia coli from wild birds in the Immediate Enviroment of Tokyo Bay. Appl Environ Microbiol. 2009;75:292-295.

Feral pigeons are synanthropic birds. In zoos, these birds invade the enclosures looking for water and food, and transmit diseases or even acquire pathogens from animals belonging to other classes, such as mammals and reptiles. The zoonotic risk associated with EPEC infection in pigeons was first documented by Silva et al. (2009) in Brazil, reporting 3.3% prevalence of infected pigeons in urban areas.1919 Silva VL, Nicoli JR, Nascimento TC, Diniz CG. Diarrheagenic Escherichia coli strains recovered from urban pigeons (Columba livia) in Brazil and their antimicrobial susceptibility patterns. Cur Microbiol. 2009;59:302-308. Population control measures in urban environments are very difficult due to the absence of natural predators. The presence of these birds in some habitats has been associated with transmission of many zoonoses such as chlamydiosis, salmonellosis and campylobacteriosis.2020 Vázquez B, Esperón F, Neves E, López J, Ballesteros C, Muñoz MJ. Screening for several potential pathogens in feral pigeons (Columba livia) in Madrid. Acta Vet Scand. 2010;52:1-6.

Sacristan et al. (2014) reported that feral pigeons from Spain were infected with atypical EPEC (8%).2121 Sacristán C, Esperón F, Herera-León S, et al. Virulence genes, antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain. Avian Pathol. 2014;43:172-175. The frequency of eae gene in urban pigeons was 6% and 4% in rural species. The authors highlighted the public health risks associated with antibiotic resistance, because some strains presented class I integrons containing genes cassetes encoding for antibiotic resistance.

We believe that the colonization of birds may vary according to the susceptibility of the species, with influence of diet, microflora and management. Apparently, the orders of Galliformes and Anseriformes are less susceptible, and the reports of diarrheagenic E. coli in these birds are rare, even in the face of many management risk factors, such as access to lakes and water collection, contact with other animals in shared enclosures and difficulty in maintaining hygiene on dirt floors.

Conclusion

This study highlights the presence of diarrheagenic E. coli (EPEC and STEC) in captive Psittaciformes, Columbiformes and Strigiformes. The zoonotic potential of these strains may be investigated because of the sanitary impact on zoo bird collections, which are important for the "in situ" conservation of the species.

Acknowledgements

The authors are thankful to Quinzinho de Barros Zoo (Sorocaba, SP - Brazil) and Parque Estoril. Zoológico Municipal de São Bernardo do Campo for fecal samples donation.

This study was conducted with financial support from CNPq and FAPESP (2014/07837-6).

References

  • 1
    Chomel BB, Belotto A, Meslin FX. Wildlife exotic pets, and emerging zoonoses. Emerg Infect Dis 2007;13:1-8.
  • 2
    Ahmed AM, Motoi Y, Sato M, et al. Zoo animals as reservoir of Gram-negative Bacteria harboring integrons and antimicrobial resistance genes. Applied Envirom Microbiol. 2007;:.
    » https://doi.org/10.1128/AEM.01054-07
  • 3
    Davies YM, Cunha MPV, Oliveira MGX, et al. Virulence and antimicrobial resistance of Klebsiella pneumoniae isolated from passerines and psittacine birds. Avian Pathol 2016;45:194-201.
  • 4
    Mattes BR, Consiglio SAS, Almeida BZ, et al. Influência da Biossegurança na colonização intestinal por Escherichia coli em psitacídeos. Arq Inst Biol 2005;72:13-16.
  • 5
    Cunha MPV, Oliveira MGX, Oliveira MC, et al. Virulence profiles, phylogenetic background and antibiotic resistance of Escherichia coli isolated from turkeys with airsacculitis. Sci World J. 2014;2014:1-8.
  • 6
    Farooq S, Hussain MA, Bhat MA, Wani SA. Isolation of atypical enteropathogenic Escherichia coli and Shiga toxin 1 and 2f-producing Escherichia coli from avian species in India. Lett Appl Microbiol. 2009;48:692-697.
  • 7
    Gioia Di-Chiacchio RM, Cunha MPV, Sturn RM, et al. Shiga toxin-producing Escherichia coli (STEC): zoonotic risks associated with psittacine pet birds in home environment. Vet Microbiol 2016;184:27-30.
  • 8
    Croxen MA, Law RJ, Scholz R, Keeney KM, Wlodarska M, Finlay BB. Recent advances in understanding enteric pathogenic Escherichia coli. Clin Microbiol Rev 2013;26:822-880.
  • 9
    Vieira MAM. Ilhas de Patogenicidade. O Mundo da Saúde. 2009;33:406-414.
  • 10
    Trabulsi LR, Keller R, Gomes TAT. Typical and atypical enteropathogenic Escherichia coli. Emerg Infect Dis 2002;8:508-513.
  • 11
    Persad AK, Lejeune JT. Animal reservoirs of Shiga toxin-producing Escherichia coli. Microbiol Spect 2014;2:1-14.
  • 12
    Costa ARF, Lima KVB, Sousa CO, Loureiro ECB. Desenvolvimento de PCR multiplex para a detecção e diferenciação de categorias de Escherichia coli diarreiogênicas. Rev Pan-Amaz Saúde 2010;1:77-84.
  • 13
    Boom R, Sol CJA, Salimans MMM. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28:453-459.
  • 14
    Kobayashi H, Kanazaki M, Hata E, Kubo M. Prevalence and characteristics of eae-and-stx-positive strains of Escherichia coli from wild birds in the Immediate Enviroment of Tokyo Bay. Appl Environ Microbiol. 2009;75:292-295.
  • 15
    Saidenberg ABS, Guedes NMR, Seixas GHF, et al. A survey for Escherichia coli virulence factors in asymptomatic free-ranging parrots. Int Schol Res Network. 2012;2012:1-6.
  • 16
    Saidenberg ABS, Teixeira RHF, Guedes NMR, Allgayer Melville PA, Benites NR. Molecular detection of enteropathogenic Escherichia coli in asyntomatic captive psittacines. Pesq Vet Bras. 2012;32:922-926.
  • 17
    Marietto-Gonçalves G, Almeida SM, Rodrigues J. Presence of a human diarrheagenic Escherichia coli clone in captivity kept Psittacidaes. Open Microbiol. 2011;5:72-75.
  • 18
    Xenoulis PG, Gray PL, Brightsmith D, et al. Molecular characterization of the cloacal microbiota of wild and captive parrots. Vet Microbiol. 2010;146:320-325.
  • 19
    Silva VL, Nicoli JR, Nascimento TC, Diniz CG. Diarrheagenic Escherichia coli strains recovered from urban pigeons (Columba livia) in Brazil and their antimicrobial susceptibility patterns. Cur Microbiol. 2009;59:302-308.
  • 20
    Vázquez B, Esperón F, Neves E, López J, Ballesteros C, Muñoz MJ. Screening for several potential pathogens in feral pigeons (Columba livia) in Madrid. Acta Vet Scand. 2010;52:1-6.
  • 21
    Sacristán C, Esperón F, Herera-León S, et al. Virulence genes, antibiotic resistance and integrons in Escherichia coli strains isolated from synanthropic birds from Spain. Avian Pathol. 2014;43:172-175.

Publication Dates

  • Publication in this collection
    Oct-Dec 2017

History

  • Received
    8 Sept 2016
  • Accepted
    8 Mar 2017
Sociedade Brasileira de Microbiologia USP - ICB III - Dep. de Microbiologia, Sociedade Brasileira de Microbiologia, Av. Prof. Lineu Prestes, 2415, Cidade Universitária, 05508-900 São Paulo, SP - Brasil, Ramal USP 7979, Tel. / Fax: (55 11) 3813-9647 ou 3037-7095 - São Paulo - SP - Brazil
E-mail: bjm@sbmicrobiologia.org.br