Abstract
Chlamydia psittaci is a zoonotic pathogen with a wide range of avian hosts and worldwide geographical spread. Zoonotic transfer occurs by inhalation or direct contact and may cause psittacosis or parrot disease. Host reservoirs of particular epidemiological interest include gregarious or migratory species, as colonial behavior facilitates microbial spread amongst conspecifics and a migratory ecology permits disperse over a wide geographic region. The current study detected C. psittaci antibodies in 76 of 81 (93.8%) feral Canada geese (Branta canadensis) using a species-specific enzyme-linked immunosorbent assay. Viable C. psittaci were isolated from pharyngeal swabs of 47 of 81 (58%) birds, and subsequent ompA-based genotyping revealed genotypes E, B, and E/B. Absence of clinical symptoms in these geese could reflect a strong natural protection or a persistent infection. Canada geese are considered an important alien species in Europe. Current results denote that this avian reservoir poses a considerable risk to native wildlife as a potential source of C. psittaci.
Introduction
Most reported psittacosis cases due to infected wild birds are on contact with feral pigeons (Magnino et al. 2009). C. psittaci–infected Canada Geese (Branta canadensis) might also present a threat to public health because they foul land and water with their droppings and respiratory mucous membrane exudate. Contaminated land and lakes or ponds are frequently visited for recreational purposes. C. psittaci remains viable at low temperatures and is resistant to desiccation. The present study examines the occurrence and genotype distribution of C. psittaci in feral Canada Geese because the project “Delivering Alien Invasive Species Inventories for Europe,” has nominated the Canada Goose as amongst the worst invasive species in Europe (Brochier et al. 2010) and current epidemiological data on C. psittaci in feral Canada Geese are lacking.
Materials and Methods
Eighty-one feral geese were manually captured and euthanized by injection of T61® (Intervet, Mechelen, Belgium) in the brachial vein as part of a governmental pest management program for controlling the fast-growing number of alien bird species in Belgium (European Interreg IV A INVEXO project). Immediately after euthanasia, pharyngeal swabs were collected and blood samples taken from the contralateral brachial vein. None of the birds showed symptoms of disease. Serum antibody titers were determined using the C. psittaci recombinant major outer membrane protein (rMOMP)-based antibody enzyme-linked immunosorbent assay (ELISA) as described by Verminnen et al. (2006). This ELISA detects antibodies against all presently known C. psittaci genotypes.
Pharyngeal swabs were examined for the presence of viable C. psittaci by inoculation on BGM cells and identification using the IMAGEN™ direct immunofluorescence assay (Oxoid, Hampshire, UK) (Vanrompay et al. 1994). A score of 0 indicates absence of chlamydial forms. Positive cultures were scored by enumeration of elementary bodies (EBs) and inclusion positive cells in five random microscopic fields (600×, Nikon Eclipse TE2000-E, Japan). Scores of 1 and 2 indicate a mean of 1–5 EBs and 6–10 EBs in the absence of inclusion positive cells, whereas scores of 3–5 were given when a mean of 1–5, 6–10, and >10 inclusion-positive cells, respectively, was present. All positive samples were passaged once, and isolates were characterized using an ompA-based, genotype-specific, real-time PCR detecting genotypes A to F and E/B (Geens et al. 2005).
Results and Discussion
The ELISA revealed C. psittaci antibodies in 76 of 81 (93.8%) clinically healthy Canada Geese. The overall mean antibody titer was 1/400. Forty-seven of 81 (58%) pharyngeal swabs were culture positive, but culture scores were low (Table 1). High prevalence of C. psittaci in feral Canada Geese as demonstrated by antibody detection in the absence of clear symptoms and an apparently low number of viable C. psittaci is suggestive of a persistent infection (Goellner et al. 2006). Genotyping was successful in 40% (19/47) of culture positive samples, revealing genotypes E, B and E/B (Table 1). All isolates were successfully passaged, yet those with a culture score of 1 at initial isolation did not yield a higher culture score after passage. EBs attached to and internalized into host cells, but replication did not occur. Insufficient amounts of DNA of those isolates may have been the underlying cause of unsuccessful genotyping.
C. psittaci–positive cells were enumerated in five randomly selected microscopy fields (600×, Nikon Eclipse TE2000-E, Japan), and results werescored from 0 to 5. Score 0 indicated that no C. psittaci was present; scores 1 and 2 were given when a mean of 1–5 and 6–10 nonreplicating elementary bodies, respectively, was present; scores 3, 4, and 5 were given when a mean of 1–5, 6–10, and >10 inclusion positive cells, respectively, was present.
Isolation of genotypes B and E may point in the direction of contact with feral pigeons, from which both genotypes are frequently isolated (Magnino et al. 2009). Infection with genotype E/B strains may rather be related to contact with waterbirds, because this genotype has so far mainly been isolated from domestic Anseriformes (Geens et al. 2005) and was recently isolated from feral Charadriiformes and Gruiiformes (unpuplished data). Transmission from or to waterbirds is also consistent with a shared ecology when Canada Geese congregate in large populations at resting and feeding sites along rivers, lakes, and seashores (Brochier et al. 2010). Stopover sites along major flyways, in particular, have been reported to connect many species and populations in time and space (Olsen et al. 2006).
Footnotes
Acknowledgments
This study was funded by the Federal Public Service of Health, Safety of the Food Chain and Environment (convention RF-6177) and by Ghent University (IOF/STEP/002). The authors kindly acknowledge K. Laroucau (Bacterial Zoonoses Unit, French Food Safety Agency, Maisons-Alfort, France) for providing control sera from ducks. A. Dumont is acknowledged for technical assistance.
Author Disclosure Statement
No competing financial interests exist.