Salmonella enterica is one of the main causes of gastrointestinal disease worldwide. Wild birds are capable of harboring a variety of Salmonella serovars, which could have an important role in the epidemiology of salmonellosis in humans and production animals. We tested 519 fecal samples from raptors and aquatic birds from different regions of central (three rehabilitation centers for wildlife and the coastal area) and southern areas of Chile for Salmonella. All samples were obtained in 2015 and 2017, covering all four seasons. Salmonella was isolated from 12 of the 519 samples (2%) analyzed, from two carnivorous birds, four birds with generalist habits, and six waterfowl. Among the isolates obtained, one showed resistance to gentamicin, and one showed a multidrug-resistance phenotype, with resistance to ampicillin, ceftriaxone, ciprofloxacin, chloramphenicol, streptomycin, gentamicin, kanamycin, trimethoprim-sulfamethoxazole, and tetracycline. These results demonstrated the importance of characterizing Salmonella in wild birds because previous studies have shown genetic and phenotypic evidence suggesting interspecies transmission of Salmonella enterica that is resistant to antimicrobials between humans and wild and domestic birds.

Salmonella enterica is one of the most relevant foodborne pathogens worldwide, with estimates of 93.8 million human cases resulting in 155,000 deaths/yr (Majowicz et al. 2010). Although production animals are considered the main reservoir and source of infection for humans, Salmonella is also present in various species of wild animals (Plym Forshell and Wierup 2006; Hoelzer et al. 2011). Wild birds have roles in the dissemination of Salmonella, including multidrug-resistant (MDR) strains (Mohsin et al. 2017). Wild birds are capable of harboring a large variety of Salmonella serovars, such as Typhimurium, Enteritidis, and Anatum (Tizard 2004; Fresno et al. 2013), including serovars that are important in human and domestic livestock salmonellosis (Navarro-Gonzalez et al. 2016). In Chile, Salmonella was reported in aquatic wild birds, with an isolation frequency in water birds, such as Kelp Gulls (Larus dominicanus) and Franklin's Gulls (Leucophaeus pipixcan) of 6–25% (López-Martín et al. 2011; Fresno et al. 2013). In those studies, Salmonella Enteritidis was the main serovar detected. To expand our knowledge of the role of wild birds as carriers of Salmonella, we aimed to determine the presence and risk factors of S. enterica in wild birds arriving at wildlife rehabilitation centers and in free-living wild birds in Chile.

This study was conducted in central and southern Chile. Samples for Salmonella isolation were obtained from birds in rehabilitation centers, wild birds, and in feces from wild birds. Three rehabilitation centers were included in the study: the Zoological Safari Park, the Metropolitan Zoo of Santiago (ZM), and the Wildlife Rehabilitation Center of Universidad Andres Bello/Buin Zoo (UFAS); all three are located in central Chile. Samples of wild birds were obtained from three locations in southern Chile, and fecal samples were obtained in the coastal city of Concon, in central Chile. In total, 519 samples were processed for Salmonella isolation; 313 samples were collected from wild birds that entered the rehabilitation centers from March 2015 to August 2016. These samples were obtained within the first 24 h of the arrival of the birds at the rehabilitation centers, before any pharmacological treatment. For samples from birds in the wild, 45 cloacal swabs were obtained in southern Chile during the months of August 2015 and April 2016 (data on the collection of these samples is available in Verdugo et al. 2019). Finally, 161 samples collected directly from fresh bird feces were obtained; these represented all fresh feces available at the sampling visit. These samples were obtained in the coastal city of Concon, which was selected because of its high diversity and confluence of wild water birds. These samples were collected from June 2017 to November 2017.

All fecal samples were collected in Cary Blair transport medium (Copan Italia Spa, Brescia, Italia) under sterile conditions and were transported within 24 h of collection at 4 C for processing at Universidad Andres Bello. For Salmonella isolation, 10 g of each sample was enriched with 90 mL of peptone water (Becton-Dickinson, Franklin Lakes, New Jersey, USA) and incubated at 37 C overnight. Subsequently, 100 µL of each sample was transferred to Rappaport Vassiliadis (Becton-Dickinson) supplemented with 20 µg/mL novobiocin (Sigma-Aldrich, St. Louis, Missouri, USA), and 1 mL of the sample was transferred on Tetrathionate (Becton-Dickinson) and incubated at 42 C. After incubation, 100 µL was transferred into agar XLT-4 (Becton-Dickinson), supplemented with sodium tetradecyl sulfate, and incubated at 37 C for 24 h. Presumptive colonies were confirmed as previously described by invA PCR (Kim et al. 2007) and stored at –80 C with glycerol.

The Kirby-Bauer technique was used to evaluate antimicrobial resistance for the following 14 antimicrobials (OXOID, Hampshire, UK): gentamicin (10 µg), streptomycin (10 µg), amoxicillin-clavulanic acid (20/10 µg), cefoxitin (30 µg), ceftiofur (30 µg), ceftriaxone (30 µg), trimethoprim-sulfamethoxazole (1.25/23.75 µg), azithromycin (15 µg), ampicillin (10 µg), chloramphenicol (30 µg), ciprofloxacin (5 µg), nalidixic acid (30 µg), tetracycline (30 µg), and kanamycin (30 µg). Escherichia coli ATCC 25922 was used as a control. Results were interpreted according to the Clinical and Laboratory Standards Institute (2017). In the Salmonella isolates, we used a molecular scheme (PCR-based) previously described to identify Salmonella serogroups B, C1, C2, D, and E1 (Ranieri et al. 2013).

A statistical analysis was conducted to determine a possible association of seasons and site with the presence of Salmonella. The variables considered for the model were site, season, and detection of Salmonella. For the site variable, only sites with positive and negative results were included (i.e., Concon, UFAS, and ZM), and sites with all negative results were excluded (i.e., samples from birds from the wild and the Zoological Safari Park). The variables, season (spring, summer, fall, and winter), dry season (spring and summer), and wet season (fall and winter) were also used, with the variable detection of Salmonella as the outcome. A model with the variables of interest was then evaluated with generalized linear model multivariable analysis to determine the association among these variables jointly and the outcome of interest. P≤0.05 was considered significant. The statistical software R Studio (version 1.1.456, RStudio, Boston, Massachusetts, USA) was used to perform all the analyses.

Among the 519 analyzed samples, 12 (2.3%) tested positive for Salmonella (Table 1). In samples from the rehabilitation centers, Salmonella was identified in 1.7% (2/121) of the birds of the ZM and in 2.2% (4/182) of the birds of the UFAS. In samples from birds in the wild, Salmonella was not detected (0/45), and in wild bird feces, 3.7% (6/161) of the Concon samples tested positive. Among the samples from the rehabilitation center, we found positive samples from carnivorous and generalist birds. Because of factors such as stress and the rehabilitation process suffered by wild animals that arrive at rehabilitation centers, there might be an increase in Salmonella shedding, so the sampling of these individuals might better reflect the prevalence of Salmonella in wildlife, in comparison with the samplings performed only on asymptomatic animals (Smith et al. 2002).

Table 1

Origin, source, number of samples, and positivity of samples obtained in this study in wild birds in Chile in 2015 and 2017.

Origin, source, number of samples, and positivity of samples obtained in this study in wild birds in Chile in 2015 and 2017.
Origin, source, number of samples, and positivity of samples obtained in this study in wild birds in Chile in 2015 and 2017.

Twelve isolates were characterized by their serogroup and antimicrobial-resistant profiles (Table 2). Most of the isolates were pansusceptible (10/12, 83%); one isolate from a carnivorous bird (Harris's hawk [Parabuteo unicinctus] obtained from the ZM) showed resistance to gentamicin, and one isolate from a generalist bird (Kelp Gull [Larus dominicanus] obtained from the UFAS) showed resistance to nine antimicrobials (ampicillin, ceftriaxone, ciprofloxacin, chloramphenicol, streptomycin, gentamicin, kanamycin, trimethoprim-sulfamethoxazole, and tetracycline). A previous study identified resistance to gentamicin, tetracycline, and ampicillin in Salmonella isolates obtained from waterfowl in Chile (Fresno et al. 2013). These results show the presence in Chile of MDR Salmonella in wild birds, which might possess a potential role as disseminators of these microorganisms and could be a reflection of the anthropogenic effect on the habitat of these birds (Mohsin et al. 2017). Within the identified serogroups, three isolates from UFAS were classified as serogroup B, and two isolates (one from ZM and one from Concon) were classified as serogroup C1; two isolates from Concon were classified as serogroup D. The other three isolates could not be classified in any of the five serogroups that we tested for. Although the Salmonella serovars most commonly isolated in wild birds of different regions of the world and Chile are Typhimurium and Enteritidis, wild birds can also harbor a great diversity of serovars not included in the serogroups tested with the scheme used here (Tizard 2004).

Table 2

Serogroup and antimicrobial resistance of isolated Salmonella from two birds at rehabilitation centers that tested positive and from samples from fresh feces from birds at Concón, Chile.

Serogroup and antimicrobial resistance of isolated Salmonella from two birds at rehabilitation centers that tested positive and from samples from fresh feces from birds at Concón, Chile.
Serogroup and antimicrobial resistance of isolated Salmonella from two birds at rehabilitation centers that tested positive and from samples from fresh feces from birds at Concón, Chile.

The multivariable model was run for the season and site variables (Table 2); this analysis showed that only the variable season had at least one level that was significant (P≤0.05). The multivariable model indicated that detecting Salmonella in birds in spring was 0.19× lower than in winter (P≤0.0471; 95% confidence interval, 0.03–0.83%). The seasonal presence of Salmonella has been previously evaluated in waterbirds in Chile (López-Martín et al. 2011); those authors also found a greater number of isolates in the winter and fall. However, the authors attributed those results to the different feeding habits of the birds (López-Martín et al. 2011). A higher frequency of Salmonella in winter was associated with wild-bird mortality events, mainly in passerine birds using garden feeders (Tizard 2004).

In our study, the variable site was not associated with Salmonella (Table 3). In other studies, fecal and anthropogenic contamination has been linked with the presence of Salmonella in wild birds, thus reflecting the role of the environment in Salmonella carriage (Tizard 2004). Interestingly, the MDR isolate found here was isolated from a Larus dominicanus from the UFAS, which presented a resistance phenotype against nine antimicrobials; MDR Salmonella isolates have been previously reported in wildlife (Molina-López et al. 2015).

Table 3

Multivariable generalized linear model showing risk-factor associations of site and season for Salmonella detection in samples from birds in Chile in 2015 and 2017.

Multivariable generalized linear model showing risk-factor associations of site and season for Salmonella detection in samples from birds in Chile in 2015 and 2017.
Multivariable generalized linear model showing risk-factor associations of site and season for Salmonella detection in samples from birds in Chile in 2015 and 2017.

Previous studies have used genetic and phenotypic evidence of isolates obtained from aquatic birds, poultry, and humans in Chile to suggest interspecies transmission of Salmonella Enteritidis (Fresno et al. 2013; Retamal et al. 2015; Toro et al. 2016). Importantly, anthropogenic contamination of the environment may facilitate infection in wild birds, which could act as an important reservoir of Salmonella Enteritidis, in which humans would be an accidental host (Toro et al. 2016). Additionally, these same factors that facilitate interspecies transmission in Salmonella Enteritidis can facilitate the transmission of antimicrobial resistance present in the Salmonella that inhabits other hosts, which are under selective pressure from contaminated environments, such as farmed animals and sewage, many of which are frequented by wild bird, such as the Kelp Gull (Retamal et al. 2015; Masarikova et al. 2016). The findings reported here contribute to our knowledge of the role of wildlife animals in the epidemiology of Salmonella with characteristics of public health interest, such as the presence of MDR.

The authors thank Isabel Campos and Carolina Sánchez from the Wildlife Rehabilitation Center (UFAS), Diego Peñaloza from the Rancagua Zoo, Carolina Ibarra and Consuelo Foerster from the Metropolitan Zoo of Santiago, and Claudio Verdugo from the Universidad Austral for their assistance in the sampling activities. Funding was from FONDECYT 11140108 to A.I.M.-S., UNAB Initiation, and the Millennium Science Initiative of the Ministry of Economy, Development and Tourism, Government of Chile.

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Author notes

4Current address: Escuela de Medicina Veterinaria, Pontificia Universidad Catœlica de Chile, Av. Vicuña Mackenna 4860, Macul, Santiago 7820436, Chile