Low occurrence of Salmonella spp. in wild birds from a Swiss rehabilitation centre

Abstract Background Salmonella are bacteria of the family Enterobacteriaceae with a wide host range. Infection in birds causes subclinical disease to mass mortality events. Wild birds may act as healthy carriers posing a hazard to livestock and humans. The present study investigated the occurrence of Salmonella in wild birds admitted to a rehabilitation centre in order to assess the exposure of the staff to this zoonotic pathogen. Methods Faecal swabs of 552 avian patients (68 species) were collected over the course of 12 months. Each sample was propagated in enrichment broth and subsequently incubated on a RAPID'Salmonella plate. Salmonella isolates were serotyped, and antimicrobial susceptibility testing was performed. Results Six Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and 1 S. Schleissheim were detected; all were pansusceptible to the antibiotics tested. Conclusion Despite the low positive rate in the tested population, the authors recommend applying protective equipment and hygiene measures when handling wild birds.


INTRODUCTION
Salmonella are Gram-negative rod-shaped bacteria belonging to the family Enterobacteriaceae. 1 The species Salmonella (S.) enterica comprises six subspecies, which are further classified into serovars (syn. serotypes) according to their O-and Hantigens (White-Kaufmann-Le Minor scheme). 2 Salmonella enterica subsp. enterica mostly occurs in warm-blooded vertebrates and currently comprises 1531 serovars.
Salmonella denote a wide host range in animals and humans, and almost all serovars have a zoonotic potential. 3 However, within some serovars, there are variants/phage types with a narrow host range, such as phage types DT2 and DT99 of Salmonella Typhimurium, which are adapted to pigeons, 4 and few serovars are strictly host-adapted, such as S. Typhi and S. Paratyphi A and C in humans. 3 Non-host adapted (ubiquitous) Salmonella usually cause self-limiting enteritis in mammals. While livestock may or may not show disease, they can become persistent shedders of Salmonella, thus promoting the spread to other animals or humans. 3 Infection in humans is generally characterized by gastrointestinal symptoms accompanied by fever, myalgias, cephalalgias and malaise over 2-4 days, and recovered patients may shed Salmonella for up to a few months, 3 again, aiding the spread of Salmonella to other individuals causing further illness and loss of revenue.
In wild birds, infection with certain Salmonella serovars, for example, Salmonella Typhimurium can lead to mass mortality events which are mainly described for passerine birds in winter. However, an infection may also take a milder clinical course or lead to clinically inapparent infection. [5][6][7] Wild birds, as very mobile species, are therefore often discussed as healthy carriers of Salmonella spp. that could pose a hazard to livestock and humans. However, it is still unclear whether wild birds function as true reservoirs or merely act as a factor in dispersal of Salmonellae acquired from the environment or livestock. 6,[8][9][10][11] Workers at wildlife rehabilitation centres present a population at risk for the transmission of zoonotic infections. Yearly between 800 and 1500 birds were admitted to the wild bird rehabilitation centre of the Swiss Ornithological Institute, Sempach, Lucerne, Switzerland during the last 10 years. Avian patients receive care (e.g., cleaning, feeding, veterinary care) depending on their condition. While manual handling is reduced to a necessary minimum, the staff of the cntre  of  Veterinary Record Open (six persons including a veterinarian) comes into close contact with the birds whenever handling is unavoidable.
The objective of the present study was to detect the Salmonella occurrence in wild birds admitted to the wild bird rehabilitation centre of the Swiss Ornithological Institute over the course of 1 year. The study aimed to assess the potential exposure of the workers at the rehabilitation centre to this zoonotic pathogen.

MATERIAL AND METHODS
For the present study, faecal swabs of the avian patients at the rehabilitation centre of the Swiss Ornithological Institute were collected over the course of 12 months, between May 2018 and May 2019, namely from 552 birds representing 68 different species of 14 orders (Table S1). For all birds, an individual animal ID, the finding place, species, age and findings from the clinical assessment were noted. Age and body condition of the birds were determined by an experienced ornithologist and veterinarian. Birds were housed and cages cleaned and disinfected as detailed by Stalder et al. 12 Swabs (Transwab Amies sterile, with Amies medium MW170; HuberLab) were taken of freshly passed faeces from a sterile surface or -in the case of dead birds -collected from the cloaca. Swabs were taken within the first 24 h after arrival of the birds at the station and prior to any treatment with antibiotics. Samples were numbered consecutively, and sample ID and sampling date were noted on the clinical record of the individual animal. Swabs were stored refrigerated for a maximum of 7 days before they were sent to the laboratory in batches.
At the laboratory, each swab was incubated in 5 ml Enterobacteriaceae Enrichment broth (BD, Franklin Lakes, USA) at 37 • C for 24 h. One loopful of each enrichment was inoculated onto RAPID'Salmonella agar (RSAL; BIO RAD Laboratories, Inc., California, USA) and incubated at 37 • C for 24 h. For confirmation of species identity, matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (Biotyper MALDI-TOF-MS, Bruker Daltonics, Billerica, MA, USA) was used. Salmonella isolates were submitted to the National Centre for Enteropathogenic Bacteria and Listeria (NENT), Switzerland, for serotyping. 2 Antimicrobial susceptibility for ampicillin, cefazolin, cefotaxime, amoxicillin-clavulanic acid, cefepim, nalidixic acid, ciprofloxacin, sulfamethoxazoletrimethoprim, fosfomycin, azythromycin, nitrofurantoin, streptomycin, kanamycin, gentamicin, chloramphenicol and tetracycline was assessed using the disk-diffusion method according to the Clinical and Laboratory Standards Institute performance standards. 13

RESULTS
Samples were unevenly distributed throughout the year corresponding to the number of birds admitted to the station, with the majority of samples taken in late spring (May/June: n = 122-146), less samples taken in early spring and summer (March/April and July/August/September: n = 36-49) and the least number of samples taken in autumn and winter (October to February: n = 4-29 per months). Accordingly, the majority of avian patients with known age (n = 497) were nestlings, pulli or hatching year birds (334, 67.2 per cent). The body condition was determined in 539 birds and was mainly good (n = 310) or moderate (n = 159) and to a lesser extent poor (n = 70). Most birds originated from the canton Lucerne (n = 400/552, 73.8 per cent), and to a lesser extent from further 14 (out of 26) Swiss cantons. All birds were found north of the alps.
Six Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and 1 S. enterica subsp. Six birds originated from Lucerne, and 1 common buzzard was found in the neighbouring canton of Obwalden (Table 1). All seven Salmonella strains were susceptible to all antibiotics tested.

DISCUSSION
Salmonella are commonly found in the intestine of wild birds. 6 Infection occurs via the oral route by ingestion of contaminated food or water, or by the consumption of infected prey. Infected birds may be asymptomatic, show various clinical signs and even develop fatal clinical disease. 6 Affected birds are often emaciated, 14 but may also die of salmonellosis in good body condition, possibly due to a short course of the disease. 6 Commonly, salmonellosis affects adult wild birds 6 or 1st-year birds after their first postjuvenile body moult, but younger birds may also be affected. 14 In this study, the body condition of birds positive for Salmonella was good for the common buzzard sampled in July and the grey heron, moderate for the passerine birds, and poor for the remaining three raptors. Both birds in good body condition were submitted with rather acute clinical signs not related to salmonellosis (Table 1) suggesting that these birds were carriers of Salmonella without being clinically affected. The three raptors with poor body condition were either found in winter (n = 2), when food for raptors is scarce, and many die due to lack of food or had a suspect history of collision trauma, possibly limiting the birds' ability to catch prey. These facts suggest that at least the first two birds were again rather carriers of Salmonella than clinically affected. However, raptors not only act as carriers of Salmonella, but may also suffer from clinical salmonellosis after feeding on infected prey species. Concomitant disease is discussed to increase the susceptibility to salmonellosis. 6 Infections with Salmonella have also been reported for various aquatic birds, 6,15-18 including an outbreak in a heron colony involving six different heron species. 19 For both Salmonella positive passerine birds, a greenfinch and a house sparrow, no clinical history or age was noted. While these species are the ones reported to be the most frequently affected by passerine salmonellosis, 7 this is often associated with multiple deaths in a small geographic area such as around bird feeders. 6

Class (Latin) Species (Latin) Species (English) Country Reference
Aves In the present study, six of seven Salmonella strains detected were identified as S. Typhimurium. Apart from certain variants / phage types, such as DT2 and DT99 in pigeons, 4 S. Typhimurium is non-host-specific as it may infect a wide array of species including humans, and it is the most common serovar reported in wild birds 6 and responsible for passerine salmonellosis. 7 Garden birds were suggested to be a reservoir for S. Typhimurium 14 and less frequently other serotypes.
S. Schleissheim on the other hand, which was detected in one common buzzard in the present study, is less frequently isolated from environmental samples, 20 mammals including humans, 21,22 birds including a Common buzzard 23 and reptiles 28 (Table 2) in different countries. While animals were not reported to show clinical signs, infection in humans was associated with enteritis. 21,22 Garden birds were suggested to be a reservoir for S. Typhimurium 14 and less frequently other serotypes.
Wild birds are often suspected to be the source of a disease incursion into farm animals, although domestic and wild birds share the same environment, and both may get infected from similar environmental sources, or pathogen transmission may consist of spill-over and spill-back events among wildlife, livestock and humans. 29 The present study investigated wild birds as possible carriers of Salmonella as a potential hazard to livestock and humans -specifically the workers of the bird rehabilitation centre. Sampling was limited to a small geographic region, due to the fact, that wild birds were not sampled in the field, but were sampled in a resource-effective manner after admittance to a rehabilitation centre. This centre, however, is located in the canton of Lucerne in the Swiss plateau, being the canton with the highest livestock density, 30 and ample opportunities for interspecies contacts are provided because of widespread free-range livestock husbandry.
Having said that, despite a non-vaccination policy in poultry and swine, the prevalence rate of Salmonella spp. in Swiss livestock is low (<2% in poultry, fattening pigs) compared to other European countries. [31][32][33] All isolated strains from animals are tested for antimicrobial susceptibility at the Swiss national reference laboratory, and the majority of Salmonella spp. isolates from cattle and poultry (2018: 62% and 83%, 2019: 76% and 78%, respectively) including all Salmonella Typhimurium isolates were susceptible to all antimicrobial classes tested. 33 The detected Salmonella isolation rate, with two different pansusceptible Salmonella enterica subsp. enterica serotypes, was low, suggesting a low hazard for livestock and also the staff of the centre to get infected. The authors nonetheless recommend wearing protective equipment and applying a stringent hygiene management whilst handling wild birds or cleaning their cages.

A C K N O W L E D G E M E N T S
We are grateful to the staff of the Swiss Ornithological Institute in Sempach, Switzerland for the collaboration, to Sandro Stalder of the NRGK for help with laboratory work, the National Reference Centre for Enteropathogenic Bacteria and Listeria for Salmonella for serotyping the strains and Roger Stephan from the Institute for Food Safety and Hygiene for critically reading the manuscript. We also thank Sonja Hartnack from the Section of Epidemiology for her consultation regarding statistical evaluations.

C O N F L I C T O F I N T E R E S T
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

E T H I C S S TAT E M E N T
This study conformed to the legal requirements of Switzerland and in accordance to the guidelines of the Swiss Ornithological Institute.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data relevant to the study are included in the article or uploaded as supplementary information.