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SHORT COMMUNICATION |
1 Departments of Infectious Diseases and
2 Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden;
3 Research Institute for Zoonotic Ecology and Epidemiology, Ölands Skogsby 6280, SE-386 93 Färjestaden, Sweden;
4 Department of Animal Ecology, Ecology Building, Lund University, SE-223 62 Lund, Sweden;
5 Department of Zoology, University of Göteborg, Box 463, SE-405 30 Göteborg, Sweden;
6 Department of Bacteriology, National Veterinary Institute, SE-751 89 Uppsala, Sweden
7 Corresponding author (email: bjornol{at}ltkalmar.se)
ABSTRACT:
Rare species with small population sizes are vulnerable to perturbations such as disease, inbreeding, or random events. The threat arising from microbial pathogens could be large and other species could act as reservoirs for pathogens. We report finding three enteric bacterial species, Salmonella Amager, Campylobacter jejuni, and urease-positive thermophilic Campylobacter, in nestling free-flying peregrine falcons (Falco peregrinus) in Sweden in 2000. Campylobacter jejuni isolates exhibited marked genetic similarities to an isolate from a human, providing a possible association between a human-associated strain of this bacterium and peregrine falcons.
Key words: Campylobacter jejuni, epidemiology, peregrine falcon, Salmonella Amager, wild birds.
Historically, peregrine falcons (Falco peregrinus) were moderately common in Sweden, with an estimated population of 900–1,400 breeding pairs (Lindberg et al., 1988). The species, being a top predator that feeds on other bird species, was extremely vulnerable to substances that bioaccumulate (i.e., mercury and organic pesticides) and that were introduced into agricultural practices in the 1940s (Lindberg, 1983). Exposure to these substances affected survival and reproduction, and in the mid 1970s, fewer than 20 breeding pairs remained in the country (Lindberg et al., 1988). A recovery program with captive breeding and release of immature birds, together with a national ban on the use of harmful chemicals, have successfully increased the wild population to almost 100 pairs. Despite this remarkable recovery, the peregrine falcon still has a vulnerable conservation status in Sweden (Gärdenfors, 2000), and small changes in fecundity and mortality rates could reduce the population again.
Infectious diseases form a potential threat to the small Swedish population. There are reports of mortality of captive falcons from Salmonella Typhimurium infection (Sykes et al., 1981; Wernery et al., 1998) and epizootics of Salmonella infection in other birds (Hurvell et al., 1974; Tauni and Österlund, 2000), but data on occurrence of pathogens in free-flying peregrine falcons are still limited (Keymer, 1972; Kirkpatrick and Trexler-Myrén, 1986). In this study, we investigated occurrence of Salmonella and Campylobacter in the Swedish peregrine falcon population and identified the isolates to species and strain level with phenotypic and genetic characterization techniques.
Fecal swabs were collected from 69 peregrine falcon nestlings during banding in a national monitoring program in 2000. Of these, 62 samples were obtained from falcons in 31 broods (mean, two nestlings per brood) from southern (n = 21 individuals from 11 broods) and northern (n = 41 individuals from 20 broods) breeding areas in Sweden. Seven additional samples were taken from captive nestlings hatched at a breeding station at Nordens Ark, Bohuslän, Sweden. The nestlings that were sampled during this investigation constituted 44% of the total number of peregrine falcons hatched in Sweden during that year.
Fecal samples were collected by sterile swabs inserted into the cloaca of the birds and were stored in transport medium with charcoal (Transwab, BioDisc, Solna, Sweden) at refrigerator temperature until analyzed. At each nest, remains of prey found in the nests were identified to species but were not analyzed for the occurrence of bacteria.
All samples were cultured by previously described procedures for detection of Campylobacter (Broman et al., 2002) and according to a routine method used by accredited Swedish clinical bacteriology laboratories to detect Salmonella from stools (Palmgren, 2002). The latter method is optimized for detection of Salmonella serovars that are pathogenic to humans. To isolate Salmonella, each sample was enriched in selenite cystine enrichment broth (Oxoid AB, Stockholm, Sweden) at 37 C for 18–24 hr before the broth was plated onto selective xylose-lysine-desoxycholate agar and incubated at 37 C for 20–24 hr. Putative Salmonella isolates were further examined by carbohydrate fermentation tests and by analyzing sensitivity to bacteriophages. The isolates were characterized according to the Kauffmann-White serotyping scheme (Kauffmann, 1972) to serovar level. To isolate thermophilic Campylobacter species, samples were plated onto selective medium (42.5 g/l Columbia Agar Base [Becton Dickinson, Cockeysville, Maryland, USA]; 5% citrated horse blood, 10 mg/l vancomycin, 2,500 IE/l polymyxin B, 5 mg/l trimethoprim) and incubated at 42 C in a microaerophilic atmosphere (85% N2, 10% CO2, 5% O2) for 72 hr, at which time the plates were examined for bacterial growth. Presumptive Campylobacter spp. were identified by limited phenotypic characterization (cell morphology; oxidase, catalase, and hippurate hydrolysis test; urease production; susceptibility to nalidixic acid).
We used two methods for a detailed genetic identification of Campylobacter isolates. First, we used a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method that produces species-specific fragment patterns of all thermophilic Campylobacter species (Fermér and Engvall, 1999). As a complement and validation of the first method, a multiplex PCR method, with primer pairs specific for Campylobacter jejuni and Campylobacter coli (Vandamme et al., 1991), also was used. In both methods, boiled lysates were used as templates for the PCR reactions.
Two Salmonella isolates were obtained from two siblings in a brood sampled in northern Sweden. These isolates were identified as Salmonella Amager.
In six (9%) of the samples, thermophilic Campylobacter spp. were isolated. Of these, two were characterized as C. jejuni by phenotypic and genotypic methods. These isolates were further investigated by macrorestriction profile (MRP) with pulsed-field gel electrophoresis and the use of SmaI and KpnI for the digestions according to a previously described protocol (Broman et al., 2002), with the exceptions that a CHEF apparatus model DR III (Bio-Rad Laboratories, Sundbyberg, Sweden) was used for electrophoresis and that gels were digitally captured by GelDoc 2000 (Bio-Rad) and optimized with QuantityOne (v.4 software, Bio-Rad). Macro restriction profiles were compared with a database of C. jejuni profiles obtained from black-headed gulls (Larus ridibundus) (n = 80), chickens (n = 50), and humans (n = 50) in the Malmö region of southern Sweden with GelCompare software (Applied Maths, Kortrijk, Belgium). They were also visually compared with an additional 172 profiles originating from a variety of wild bird species and with 49 human isolates obtained in Kalmar county, southern Sweden. The SmaI and KpnI MRP of the falcon isolates were identical to each other and to the profile of a C. jejuni isolated from a young man from southern Sweden in the summer of 1999 (Fig. 1
).
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Campylobacter jejuni is commonly found in many bird species (e.g., Broman et al., 2002; Waldenström et al., 2002). However, finding that the isolates, after digestion with the enzymes (SmaI and KpnI), were genetically indistinguishable from a human clinical isolate is noteworthy. Campylobacter jejuni is a heterogeneous organism, exhibiting a multitude of genotypes. We have hundreds of MRP from wild birds and from human isolates in our data bank, and only occasionally do we find complete matches between isolates from the two hosts (Broman et al., 2002). The findings reported here, therefore, indicate that a bacterial pathogen that causes disease in humans is found in peregrine falcons. This pathogen could be from a source common to both species. Considering the diet of peregrine falcons, with a large proportion of gulls, pigeons, and corvids, one likely pathway could be consumption of a prey species that also occurs in urban areas.
Campylobacter bacteria belonging to the UPTC group, by some authors considered a biovar of C. lari (Vandamme et al., 1991), have been isolated from gulls (Kaneko et al., 1999), shellfish (Wilson and Moore, 1996), river water (Bolton et al., 1987), seawater (Bolton et al., 1985), and a few humans (Mégraud et al., 1988). Even though there seems to be an aquatic link with many of these isolates, it is unlikely that UPTCs are environmental organisms. Their optimal growth temperature of 42 C indicates that they might only multiply well in warm-blooded hosts, most likely birds, because they typically have higher body temperatures than mammals.
We do not know whether the bacteria found in these birds are pathogenic to their host. The UPTCs are generally considered to be of low pathogenicity in humans, but no work regarding pathogenicity has been performed in wild birds. Campylobacter jejuni, which is a pathogen in humans, is often regarded as nonpathogenic in birds, but even though older birds seem to be unaffected by colonization, there are reports of enteric disease and mortality in young broiler chickens because of C. jejuni infection. To our knowledge, only one published investigation has dealt with this question in a wild bird species. Twenty-seven trapped herring gulls (Larus argentatus) that were naturally colonized by C. jejuni were caged and observed for signs of disease. All birds appeared healthy, and the infection was eliminated in all but one individual at resampling 4 wk later (Glünder et al., 1992). It is important to note, though, that in a strongly heterogeneous organism like C. jejuni, virulence properties can differ between isolates. Also, it is not unlikely that host species differ in their susceptibility to infection and in severity and type of clinical manifestations.
In the case of the Salmonella Amager isolates, no direct link could be established between the falcons and other sources. We noted, however, remains of several black-headed gulls, a scavenging bird species often associated with human activities, at the infected nest, and in a previous year, remains of spaghetti have been found at the same nest.
The occurrence of salmonellosis in Sweden is extremely low. In 2000, fewer than 4,850 human cases were reported in a population of nearly 9 million. Of these, only 691 human cases were considered domestically acquired. Prevalence of Salmonella infections in domestic animals reported in 2000 was less than 0.1% (Swedish National Veterinary Institute, 2001). Except for the two falcon isolates in this study, no Salmonella Amager was reported in Sweden in the year 2000, and only one or two isolates of Salmonella Amager had been reported each year during the last 10 yr. This bacterium has been described in a few outbreaks in humans in Russia and India (Beliakov et al., 1969; Oak et al., 1984), and all known human cases of Salmonella Amager infection in Sweden were associated with travel to southern Europe or Africa. No reservoirs for this bacterium are known from Sweden (Swedish Institute for Infectious Disease Control, R. Wollin, pers. comm.).
All infected peregrine nestlings in this study fledged, but there is no other information on their further development and survival. Thus, we have no information about whether the birds in which Salmonella or Campylobacter were detected were detrimentally affected by their infections. Nevertheless, finding Salmonella Amager and C. jejuni in peregrine falcons is noteworthy and shows that this rare falcon species is exposed to enteropathogens. Further work is needed to establish the source of these infections.
This study was done in cooperation with the Peregrine Project conducted by the Swedish Society for Nature Conservation. S. Hamrén, C. Larsson, L. Nilsson, A. Wedman, J. Ekenstedt, and the late J. Engström helped us with the sampling. Many thanks to P. D. Haemig for valuable comments on the manuscript. This work was supported by the Health Research Council of Southeast Sweden (2001–02), the Center for Environmental Research, the Medical Faculty of Umeå University, and the Swedish Society of Medicine.
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Received for publication 22 May 2003.
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