An Atypical Enteropathogenic Escherichia coli Isolate with Possible Human and Domestic Animal Origin from a Free-Living Lear's Macaw (Anodorhynchus leari)

Parrots (Psittaciformes) are among the most-endangered species on earth, and macaws (Family Psittacidae, subfamily Arinae, tribe Arini) are the most affected group (IUCN 2016). Lear's Macaw (or Indigo Macaw; Anodorhynchus leari) is classified as endangered due to threats affecting its habitat and trapping for illegal trade, and little is known about its health status in the wild (Birdlife International 2016). Health surveys of free-ranging parrots are scarce and are important in generating knowledge to promote better conservation measures for species in nature and for developing captive breeding programs.

A parrot's microbiota mainly consists of Gram-positive bacteria, and the presence of Gram-negative bacteria, such as Escherichia coli, has been reported in captivity as a sign of deficient husbandry that may cause diseases ranging from respiratory conditions to diarrhea and sepsis (Bangert et al. 1988; Gerlach 1994). Enteropathogenic E. coli (EPEC) is an important pathotype causing disease in humans and other animals (Trabulsi et al. 2002). There are reports of EPEC in captive psittacines, and outbreaks may cause increased mortality if treatment and prevention measures are not applied (Seeley et al. 2014).

To improve knowledge of the pathogenic potential of microorganisms that may affect an endangered population, we conducted health surveys of Lear's Macaw to identify the prevalence of several pathogens. During these surveys, an atypical EPEC with pathogenic potential was isolated and phylogenetically characterized.

Fifty wild nestlings, showing no signs of diarrhea or abnormal development, were sampled in Canudos, Brazil (09°53′48′′S, 39°01′35′′W). Nest cavities were accessed by rappelling from February 2011–April 2014, and nestlings were manually restrained and cloacal swabs collected (permits Sistema de Autorizacao e Informacao em Biodiversidade 12763-4) using standard sampling techniques. Swabs were placed in transport medium (BD^® Diagnostic Systems, Sparks, Maryland, USA), maintained at room temperature in the field, and then refrigerated (10 C) pending laboratory analysis (<2 mo postcollection).

Each swab was processed for aerobic culture and identified using standard biochemical tests. A loop of each E. coli isolate was suspended in 200 μL of saline solution and DNA was extracted following Boom et al. (1990). We performed PCR to detect two genes characterizing EPEC; attaching and effacing (eae) and bundle-forming pili (bfp), using described primers and PCR conditions (Aranda et al. 2007). The reference EPEC strain E2348/69 and water served as positive and negative controls, respectively.

We purified the eae amplicon using GFX PCR DNA and the gel band purification kit (GE Healthcare^®, Little Chalfont, Buckinghamshire, UK) and sequenced DNA using an ABI-377 (Applied Biosystems^®, Foster City, California, USA). The sequence was submitted to GenBank (accession KF032630).

Similar sequences available in GenBank were aligned using the ClustalW tool of Bioedit (Hall 1999). The phylogenetic tree based on the partial eae gene (918 base pairs) was constructed with Mega software (Tamura et al. 2011) using neighbor joining with 1,000 bootstrap repetitions and maximum composite likelihood methods (Fig. 1).

Thirty-two sampled individuals (64%) were positive for E. coli growth. One sample from an asymptomatic chick was positive for the eae gene but negative for bfp, leading to characterization as an atypical EPEC. The phylogeny of the isolate showed a high degree of identity with GenBank sequences from humans and livestock and from urban birds that died from unknown causes. The isolate grouped in a cluster 100% identical to sequences of bovine and human isolates from several countries. It also had 96.2–99.5% identity with the sequences of other groups including 98.9% with a human EPEC infection in Brazil.

The EPEC pathotype has been described to cause disease affecting parrots and other birds (Foster et al. 1998; Schremmer et al. 1999). The typical EPEC reservoir is humans, but atypical EPEC is found in humans and other animals. Although they differ in the characteristics of adherence patterns to the intestinal epithelium, both may cause disease that results in attaching and effacing lesions (Hernandes et al. 2009).

Lear's Macaw inhabits an isolated area; however, birds fly out daily to forage for food. Foraging locations are often close to human habitations; in some instances, birds will feed on predigested palm nuts excreted by domestic goats. This behavior could lead to contact between potential pathogens shared between livestock and humans because sanitation and animal husbandry conditions are rudimentary and do not follow standards to prevent contamination of the environment and thus disease spread.

Because the isolate's sequence was identical to strains obtained from bovine and human isolates, and included high identity to a human sequence described in Brazil, there is potential for transmission between livestock and humans and spillover to wildlife.

The route of transmission could be linked to the parents feeding on the predigested nuts and regurgitating to the nestling. Because we cannot sample adults due to the risks of capture, this hypothesis is difficult to test. Whether the isolate was transient or was colonizing the intestines is unknown. The sibling in the same nest was not EPEC positive, although it had E. coli growth, and both chicks fledged successfully. During a study of reproductive biology of Lear's Macaw, Assis (2012) reported neonatal mortality, but the samples were too deteriorated to differentiate between pathogens or natural events such as hatching asynchrony.

Although only one EPEC was obtained, the risk of disease may be higher when birds are subjected to stress. This was observed in captive parrots where clinical signs of severe diarrhea and sepsis were reported with EPEC infection, resulting in outbreaks (Knöbl et al. 2011; Seeley et al. 2014). In a population that faces pressure from poaching and climate change, this could lead to further challenges to recovery of this endangered species.

Surveys of recently deceased nestlings of the Lear's macaw, as well as domestic animals in the foraging areas, might help better define the risk for EPEC transmission and the significance of this pathogen for species conservation.

We are grateful for the assistance of E. C. P. Assis and the Lear's Macaw Project, the funds from the World Parrot Trust, and the scholarships provided by Fundacao de Amparo a Pesquisa do Estado de São Paulo (10/51015-0 and 11/50375-5) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.