Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 2002 Nov;68(11):5600–5606. doi: 10.1128/AEM.68.11.5600-5606.2002

Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Isolates Determined by Pulsed-Field Gel Electrophoresis: Comparison of Isolates from Avian Wildlife, Domestic Animals, and the Environment in Norway

Thorbjørn Refsum 1,*, Even Heir 2, Georg Kapperud 2,3, Traute Vardund 2, Gudmund Holstad 1
PMCID: PMC129883  PMID: 12406755

Abstract

The molecular epidemiology of 142 isolates of Salmonella enterica serovar Typhimurium from avian wildlife, domestic animals, and the environment in Norway was investigated using pulsed-field gel electrophoresis (PFGE) and computerized numerical analysis of the data. The bacterial isolates comprised 79 isolates from wild-living birds, including 46 small passerines and 26 gulls, and 63 isolates of nonavian origin, including 50 domestic animals and 13 environmental samples. Thirteen main clusters were discernible at the 90% similarity level. Most of the isolates (83%) were grouped into three main clusters. These were further divided into 20 subclusters at the 95% similarity level. Isolates from passerines, gulls, and pigeons dominated within five subclusters, whereas isolates from domestic animals and the environment belonged to many different subclusters with no predominance. The results support earlier results that passerines constitute an important source of infection to humans in Norway, whereas it is suggested that gulls and pigeons, based on PFGE analysis, represent only a minor source of human serovar Typhimurium infections. Passerines, gulls, and pigeons may also constitute a source of infection of domestic animals and feed plants or vice versa. Three isolates from cattle and a grain source, of which two were multiresistant, were confirmed as serovar Typhimurium phage type DT 104. These represent the first reported phage type DT 104 isolates from other sources than humans in Norway.

There is strong evidence that Salmonella enterica serovar Typhimurium has established reservoirs in wild-living birds and hedgehogs in Norway (15, 27). Wild-living birds and hedgehogs may function as effective spreaders of Salmonella bacteria to humans and to different animal species through contamination of the environment (15, 23). In Norway, sporadic indigenous cases and a national outbreak of human salmonellosis, caused by serovar Typhimurium, have been related to infections in small passerines (22, 23). In a waterborne serovar Typhimurium infection outbreak in northwestern Norway in 1999, gulls were suggested to be the most likely source of infection (1; T. Refsum, G. Kapperud, and G. Holstad, submitted for publication). Moreover, two human outbreaks in 1996 and 2000 have been associated with infected hedgehog populations (15). It is therefore important to gain further knowledge of the epidemiology of Salmonella bacteria in wild-living species, in particular of the endemically distributed serovar Typhimurium.

Most previous studies are based on analytical epidemiological investigations and traditional phenotypic analysis, such as biotyping, serotyping, antimicrobial susceptibility, and phage typing. However, the limitations associated with several of these techniques have stimulated interest in DNA-based typing methods, such as pulsed-field gel electrophoresis (PFGE) (3, 5). This method has proven to be highly discriminatory and comparable or superior to other techniques (3) and has been useful in epidemiological investigations of, e.g., serovar Typhimurium outbreaks (5, 26, 31). In Norway, several serovar Typhimurium outbreaks in humans have been investigated by this method (16). The PFGE method might also be useful in surveillance of variants of particular interest, such as the multiresistant serovar Typhimurium definitive phage type DT 104. Fingerprinting by PFGE may be a valuable tool in epidemiological investigation and surveillance by relating isolates from different sources to a common origin.

The aim of this study was to investigate the molecular epidemiology of serovar Typhimurium isolates from wild-living birds, domestic animals, and the environment by using genomic fingerprinting by PFGE.

MATERIALS AND METHODS

Isolate characteristics.

A total of 142 isolates of serovar Typhimurium from avian wildlife, domestic animals, and other sources in Norway were investigated (Table 1). The 79 isolates from wild-living birds comprised 46 isolates of serovar Typhimurium O:4,12 randomly selected from a total of 350 small passerine isolates, using a random-number generator (9), and all available isolates of the serovar Typhimurium variants O:4,12 and O:4,5,12 from other wild-living bird species. The isolates were obtained from birds from all over the country. All isolates originated from birds received for postmortem examination at the National Veterinary Institute during 1969 to 2000 (27) or from surveys of wild-living birds conducted during 1997 to 2001 (T. Refsum, T. Vikøren, K. Handeland, G. Holstad, and G. Kapperud, submitted for publication; Refsum et al., submitted), except for two isolates from a crow and a gull collected in a follow-up investigation of a human outbreak in southeastern Norway in 1996 (15).

TABLE 1.

The 142 isolates of serovar Typhimurium from avian wildlife, domestic animals, and the environment according to origin, year of isolation, county, and PFGE clustera

Origin Yr of isolation Serotype County PFGE cluster
Black-headed gull, Larus ridibundus 1994 O:4,5,12 Hedmark A1
Dairy cattle 1995 O:4,5,12 Rogaland A1
Dairy cattle 1997 O:4,5,12 Rogaland A1
City pigeon, Columba livia 1993 O:4,12 Hordaland A1
City pigeon, Columba livia 1993 O:4,5,12 Oslo A1
Domestic pigeon 1994 O:4,12 Møre og Romsdal A1
Domestic pigeon 1994 O:4,12 Rogaland A1
Domestic pigeon 1994 O:4,12 Rogaland A1
Domestic pigeon 1995 O:4,12 Rogaland A1
Domestic pigeon 1998 O:4,12 Hedmark A1
Domestic pigeon 1999 O:4,12 Hordaland A1
Domestic pigeon 2000 O:4,12 Rogaland A1
Feed plant 1995 O:4,12 Østfold A1
Fish feed 1994 O:4,12 Rogaland A1
Horse 1994 O:4,5,12 Vestfold A1
Horse 1994 O:4,5,12 Oslo A1
Swine 1994 O:4,5,12 Hedmark A1
Swine 1994 O:4,5,12 Rogaland A1
Swine feed 1994 O:4,5,12 Oslo A1
Bullfinch, Pyrrhula pyrrhula 1969 O:4,12 Oslo A2
Bullfinch, Pyrrhula pyrrhula 1972 O:4,12 Vest-Agder A2
Bullfinch, Pyrrhula pyrrhula 1982 O:4,12 Oppland A2
Bullfinch, Pyrrhula pyrrhula 1982 O:4,12 Nord-Trøndelag A2
Bullfinch, Pyrrhula pyrrhula 1988 O:4,12 Oslo A2
Bullfinch, Pyrrhula pyrrhula 1988 O:4,12 Møre og Romsdal A2
Bullfinch, Pyrrhula pyrrhula 1992 O:4,12 Oslo A2
Bullfinch, Pyrrhula pyrrhula 1999 O:4,12 Østfold A2
Bullfinch, Pyrrhula pyrrhula 1999 O:4,12 Nordland A2
Bullfinch, Pyrrhula pyrrhula 1999 O:4,12 Nord-Trøndelag A2
Bullfinch, Pyrrhula pyrrhula 2000 O:4,12 Nordland A2
Bullfinch, Pyrrhula pyrrhula 2000 O:4,12 Troms A2
Cat 1998 O:4,12 Østfold A2
Cat 2000 O:4,12 Nordland A2
Dairy cattle 1995 O:4,12 Rogaland A2
Common redpoll, Carduelis flammea 2000 O:4,12 Nordland A2
Eurasian siskin, Carduelis spinus 1974 O:4,12 Akershus A2
Eurasian siskin, Carduelis spinus 1999 O:4,12 Hedmark A2
Eurasian siskin, Carduelis spinus 1999 O:4,12 Telemark A2
Fur animal feed 1997 O:4,12 Unknown A2
Geese, breeding 1995 O:4,12 Østfold A2
Great tit, Parus major 1975 O:4,12 Akershus A2
Great tit, Parus major 1994 O:4,12 Vest-Agder A2
Greenfinch, Carduelis chloris 1969 O:4,12 Oslo A2
Greenfinch, Carduelis chloris 1969 O:4,12 Vestfold A2
Greenfinch, Carduelis chloris 1969 O:4,12 Oslo A2
Greenfinch, Carduelis chloris 1972 O:4,12 Østfold A2
Greenfinch, Carduelis chloris 1972 O:4,12 Akershus A2
Eurasian tree sparrow, Passer montanus 1982 O:4,12 Akershus A2
Poultry 1995 O:4,12 Rogaland A2
Poultry 1995 O:4,12 Oppland A2
Swine 1995 O:4,12 Rogaland A2
Swine 1995 O:4,12 Østfold A2
Swine 1995 O:4,12 Østfold A2
Swine 1996 O:4,12 Rogaland A2
Swine 1999 O:4,12 Østfold A2
Black-headed gull, Larus ridibundus 1977 O:4,12 Rogaland B1
Bullfinch, Pyrrhula pyrrhula 1972 O:4,12 Oslo B1
Canada goose, Branta canadensis 1989 O:4,12 Oslo B1
Common guillemot, Uria aalge 1997 O:4,12 Oslo B1
Mew gull, Larus canis 1997 O:4,12 Oslo B1
Mew gull, Larus canis 2000 O:4,12 Hordaland B1
Mew gull, Larus canis 2001 O:4,12 Hordaland B1
Feed plant 1998 O:4,12 Vestfold B1
Feed plant 1995 O:4,5,12 Oslo B1
Great black-backed gull, Larus marinus 2001 O:4,5,12 Møre og Romsdal B1
Herring gull, Larus argentatus 1999 O:4,12 Nordland B1
Herring gull, Larus argentatus 2000 O:4,5,12 Oslo B1
Herring gull, Larus argentatus 2000 O:4,12 Hordaland B1
Herring gull, Larus argentatus 2000 O:4,12 Buskerud B1
Duck, breeding 1995 O:4,5,12 Telemark B2
Abattoir 1999 O:4,12 Hedmark B3/PICK>
Blue tit, Cyanistes caeruleus 1989 O:4,12 Hordaland B3
Bullfinch, Pyrrhula pyrrhula 1972 O:4,12 Hedmark B3
Bullfinch, Pyrrhula pyrrhula 1972 O:4,12 Akershus B3
Bullfinch, Pyrrhula pyrrhula 1982 O:4,12 Nordland B3
Bullfinch, Pyrrhula pyrrhula 1982 O:4,12 Nordland B3
Bullfinch, Pyrrhula pyrrhula 1988 O:4,12 Telemark B3
Bullfinch, Pyrrhula pyrrhula 1990 O:4,12 Nordland B3
Bullfinch, Pyrrhula pyrrhula 1998 O:4,12 Vest-Agder B3
Bullfinch, Pyrrhula pyrrhula 1998 O:4,12 Sør-Trøndelag B3
Bullfinch, Pyrrhula pyrrhula 1999 O:4,12 Buskerud B3
Cat 1998 O:4,12 Nord-Trøndelag B3
Cat 2000 O:4,12 Rogaland B3
Dairy cattle 2000 O:4,12 Sør-Trøndelag B3
Common redpoll, Carduelis flammea 1999 O:4,12 Sør-Trøndelag B3
Eurasian siskin, Carduelis spinus 1999 O:4,12 Nord-Trøndelag B3
Eurasian siskin, Carduelis spinus 1999 O:4,12 Oslo B3
Eurasian siskin, Carduelis spinus 1999 O:4,12 Sogn og Fjordane B3
Eurasian siskin, Carduelis spinus 1999 O:4,12 Østfold B3
Great tit, Parus major 1988 O:4,12 Oslo B3
House sparrow, Passer domesticus 1999 O:4,12 Nord-Trøndelag B3
House sparrow, Passer domesticus 2000 O:4,12 Nord-Trøndelag B3
Red fox 1995 O:4,12 Oppland B3
Greenfinch, Carduelis chloris 1969 O:4,12 Oslo C
Dairy cattle 1991 O:4,12 Buskerud D
Bullfinch, Pyrrhula pyrrhula 1971 O:4,12 Hedmark E
Turkey 2000 O:4,12 Østfold F
Black-headed gull, Larus ridibundus 1984 O:4,5,12 Sør-Trøndelag G1
Black-headed gull, Larus ridibundus 1993 O:4,5,12 Oslo G1
Black-headed gull, Larus ridibundus 1994 O:4,5,12 Hedmark G1
Black-headed gull, Larus ridibundus 1997 O:4,12 Oslo G1
Dairy cattle 1995 O:4,5,12 Nord-Trøndelag G1
Dairy cattle 1996 O:4,5,12 Vest-Agder G1
Dairy cattle 1997 O:4,5,12 Østfold G1
Dairy cattle 1999 O:4,5,12 Rogaland G1
Dairy cattleb 1992 O:4,12 Hordaland G1
City pigeon, Columbia livia 1993 O:4,5,12 Oslo G1
Mew gull, Larus canis 1997 O:4,5,12 Oslo G1
Mew gull, Larus canis 2000 O:4,5,12 Hordaland G1
Feed plant 1997 O:4,5,12 Østfold G1
Great black-backed gull, Larus marinus 2000 O:4,5,12 Hordaland G1
Herring gull, Larus argentatus 1999 O:4,5,12 Sogn og Fjordane G1
Herring gull, Larus argentatus 2000 O:4,5,12 Hordaland G1
Herring gull, Larus argentatus 2000 O:4,5,12 Hordaland G1
Herring gull, Larus argentatus 2000 O:4,5,12 Akershus G1
Horse 1999 O:4,5,12 Rogaland G1
Horse 2000 O:4,12 Sør-Trøndelag G1
Lesser black-backed gull, Larus fuscus 2000 O:4,5,12 Oslo G1
Magpie, Pica pica 1993 O:4,5,12 Oslo G1
Passerine birdb 1992 O:4,12 Hordaland G1
Swine 2000 O:4,12 Nordland G1
Domestic pigeon 1999 O:4,12 Aust-Agder G2
Feed plant 1997 O:4,12 Vestfold H1
Wheat-based feed 1995 O:4,5,12 Unknown H1
Domestic pigeon 2001 O:4,12 Hordaland H2
Domestic pigeon 2001 O:4,12 Sogn og Fjordane H2
Dairy cattle 1997 O:4,5,12 Møre og Romsdal H3
Dairy cattle 2001 O:4,5,12 Rogaland H3c
Grain for human consumption 1997 O:4,5,12 Oslo H3c
Ostrich 1996 O:4,5,12 Hordaland I
Dairy cattle 1996 O:4,5,12 Hordaland J1
Gull feathers sample from water supply 1999 O:4,5,12 Møre og Romsdal J1
Herring gull, Larus argentatus (adult) 1999 O:4,5,12 Møre og Romsdal J1
Herring gull, Larus argentatus (chick) 1999 O:4,5,12 Møre og Romsdal J1
Herring gull, Larus argentatus (chick) 1999 O:4,5,12 Møre og Romsdal J1
Sheep 1998 O:4,5,12 Hordaland J2
Gull, unknown species 1997 O:4,5,12 Østfold K
Hooded crow, Corvus corone 1997 O:4,5,12 Østfold K
Poultry 1993 O:4,5,12 Akershus L
Swine 1994 O:4,5,12 Oppland L
Swine 1995 O:4,5,12 Hedmark L
Feed plant 1998 O:4,5,12 Oslo M
Open in a new tab
a

The genus and species of the wild-living birds are specified. The cluster capital letters and subcluster suffixes correspond to the dendrogram in Fig. 1.

b

Serovar Typhimurium was isolated from cattle and from a small passerine bird found dead within the same cowshed.

c

Serovar Typhimurium DT 104 isolate, multiresistant to ACSSuT.

A total of 63 isolates of serovar Typhimurium isolated from sources other than wild-living birds from 1990 to 2001 were included (Table 1). All but four of the nonavian isolates originated from southern Norway. All available epidemiologically unrelated isolates from domestic animals were examined. If more than one isolate was obtained from the same herd or production unit, only one of the isolates was included. The total number comprised 50 isolates from cattle (n = 13), swine (n = 10), horses (n = 4), cats (n = 4), sheep (n = 1), poultry (n = 3), waterfowl (n = 2), turkey (n = 1), ostrich (n = 1), red fox (n = 1), and domestic pigeon (n = 10) (Table 1). In addition, 13 environmental isolates, recovered from a contaminated water supply (n = 1), from an abattoir (n = 1), and from feed (n = 10) and food (n = 1) plants were investigated. The isolates were obtained from the National Veterinary Institute and the National Salmonella Reference Laboratory at the Norwegian Institute of Public Health.

Phenotypic and genotypic characterization.

All isolates included in the present study were analyzed by PFGE. DNA preparation, restriction enzyme digestion (XbaI), and PFGE were performed as previously described (17). Lambda DNA (Sigma, St. Louis, Mo.) served as a molecular size standard in all PFGE investigations. After electrophoresis, PFGE gels were stained with ethidium bromide and photographed with GelDoc 2000 using Quantity One software (Bio-Rad, Hercules, Calif.). Thirty-one of the passerine isolates included in our study have previously been phage typed (27) by the typing scheme of Callow (8), as extended by Anderson et al. (2). In addition, three isolates from cattle and a grain source, which showed a PFGE profile characteristic for phage type DT 104 (16), were phage typed and subjected to antibiotic susceptibility testing as described by Leegaard et al. (24).

Computerized numerical analysis of PFGE data.

Images saved in TIFF format were transferred to the GelCompar II software (Applied Maths, Kortrijk, Belgium) for computer analysis. Similarity between fingerprints was determined on the basis of the Dice coefficient. A band position tolerance of 2% was used for analysis of PFGE patterns. Dendrograms were generated by the unweighted pair group method with arithmetic averages. Capital letters (A to M) were used to designate the main cluster lineages of serovar Typhimurium isolates in the dendrogram, while subclusters were given numerical suffixes.

RESULTS

In our study, 13 main clusters (designated A to M) were discernible at the 90% similarity level (Fig. 1). Most of the isolates (83%) were grouped into three main clusters: A (39%; n = 55) B (27%; n = 38), and G (18%; n = 25) (Fig. 1; Table 1). The main clusters could be further divided into 20 subclusters at the 95% similarity level. Most of the wild-bird isolates (90%) belonged to only five of these subclusters (Fig. 1 and 2), whereas isolates from domestic animals and other sources belonged to many different subclusters, with no predominance of isolates in distinct subclusters.

FIG. 1.

FIG. 1.

Open in a new tab

Dendrogram based on PFGE macrorestriction profiles of 142 isolates from avian wildlife, domestic animals, and the environment in Norway. The 13 clusters (90% similarity level) are designated by capital letters (A to M), while the subclusters (95% similarity level) are assigned numerical suffixes.

FIG. 2.

FIG. 2.

Open in a new tab

PFGE of serovar Typhimurium isolates from avian wildlife in Norway. Representatives of the five most prevalent subclusters and typical origin are shown. Lanes: 1 and 7, molecular weight marker, lambda ladder; 2, A1 (pigeon); 3, A2 (small passerine); 4, B1 (gull); 5, B3 (small passerine); 6, G1 (gull).

The A2 and B3 subclusters comprised 42 (91%) of the passerine isolates. No isolates from other wild-bird species belonged to these subclusters. Subcluster A2 also harbored 12 isolates from swine, poultry, cats, cows, breeding geese, and fur animal feed, whereas B3 contained five isolates originating from domestic cats, cows, red foxes, and an abattoir. All isolates within subclusters A2 and B3 belonged to the antigenic variant serovar Typhimurium O:4,12. Both subclusters contained passerine isolates originating from all over the country. Of the 31 phage-typed passerine isolates, all 16 isolates belonging to phage type DT 40 fell into subcluster A2, whereas all 12 isolates of phage type U277 fell into subcluster B3 together with two DT 99 isolates and one isolate classified as RDNC (routine dilution no conformity).

In parallel to passerine bird isolates, 20 (77%) of the isolates from gulls belonged to two subclusters, B1 and G1. Subcluster B1 was made up predominantly of isolates from gulls (n = 9) although sporadic isolates from other wild-living birds and two feed plants also fell into this subcluster. Subcluster G1 included 12 isolates from gulls in addition to 13 isolates from other wild-living birds, cattle, horse, swine, and a feed plant. Both subcluster B1 and G1 clones were geographically widespread.

The A1 subcluster (n = 19) was made up predominantly of isolates from pigeons, including seven domestic pigeons and two wild-living city pigeons. This subcluster also included isolates from horses, cattle, swine, a black-headed gull, and animal feed plants. Both antigenic variants of serovar Typhimurium were represented in the cluster. All the A1 isolates originated from southern Norway.

The other main clusters (C to F and H to M) were composed of the remaining 24 isolates (17%) (Table 1). The three isolates within subcluster H3, which originated from cattle and from grain meant for human consumption, showed a PFGE pattern previously found to be characteristic of phage type DT 104 (15). Phage typing confirmed that they belonged to this type, and two of them expressed a multiresistant pattern typical for DT 104 strains (ACSSuT: resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline).

Subcluster J1 contained isolates from gulls and a gull feather sample collected from a contaminated surface drinking-water supply in northwestern Norway, as well as an isolate from a cow in western Norway. Two isolates from a gull and a crow in southeastern Norway fell into subcluster K. Subcluster L comprised two isolates from swine and one isolate from poultry. This subcluster PFGE L pattern was not obtained from any of the isolates from wild birds.

DISCUSSION

The high similarity found between isolates from wild-living birds indicates a close genetic relationship between avian serovar Typhimurium isolates compared to that of isolates from humans and other sources. Previous investigations reporting clonal relationships of serovar Typhimurium from humans (16) and various other sources (26) showed that isolates of this serovariant were clustered within a window of similarity of 70%. In spite of the tight genetic relationship, it was possible to divide the majority of the wild avian isolates into five subclusters. With four exceptions, isolates from small passerines, gulls, and pigeons fell into different subclusters. The PFGE results thus indicate little crossover of Salmonella bacteria among those birds. However, it is also possible that this observation represents different host preferences of the genotypes. The three subclusters made up predominantly of isolates from gulls and pigeons also contained isolates from other wild-living bird species. It is impossible to determine whether or not this represents transmission from gull and pigeon reservoirs.

Previous investigations including a case-control study and plasmid profile and phage type analyses (21, 22, 23) have shown an epidemiological link between small passerines and humans. Recently, Heir et al. (16) have estimated that the clones within subclusters A2 and B3 (designated F1 and F3, respectively, by Heir et al.) were responsible for 32% of the sporadic, domestic human cases of serovar Typhimurium O:4,12 infection during 1996 to 1999. Thus, our PFGE results reinforce previous investigations suggesting that wild passerine birds are an important source of human serovar Typhimurium infections.

The pigeon patterns, of which the majority belonged to the heterogeneous A1 subcluster, were detected only twice in strains isolated from Norwegian patients by Heir et al. (16) (designated F9). Their study included a representative collection of 102 isolates of both foreign and domestic origins. Our results might thus support previous studies based on biochemical and phage type analyses, which suggest that the zoonotic infection hazard is of limited significance (14, 28, 33, 36, 37).

The most common gull profiles (B1 and G1) were also seen only twice among the human isolates studied by Heir et al. (designated F6 and L1, respectively, by Heir et al.) (16). It has been suggested that Salmonella-carrying gulls are of little significance as a health hazard to domestic animals and humans (11, 13). Nevertheless, gulls washing and roosting in drinking-water supplies constitute a potential human health hazard, especially if the water is consumed without prior disinfection (6, 12). Gulls were considered the most likely source of infection in a waterborne human outbreak in northwestern Norway in 1999 (1). In the present study, isolates from gulls, sampled in the municipality where the outbreak occurred, were found to be identical to the outbreak clone (J1) (Fig. 1; Table 1). However, in the follow-up survey of gulls along the Norwegian coast (Refsum, Holstad, and Kapperud, submitted), we failed to detect this clone elsewhere.

Among sporadic human isolates, the J1 profile (termed E5 by Heir et al. [16]) was observed throughout 1996 to 1999 but was geographically restricted to only two counties in western Norway. Both the J1 and L clones were implicated in an outbreak in Bergen and in two neighboring municipalities during autumn 2000 (16, 30). The L clone caused previous outbreak in southeastern Norway in 1996 (16, 34). Based on findings of the J1 and K clones in extensively infected hedgehog populations within the areas of the outbreaks in 1996 and 2000 (15), one might suggest that hedgehogs constitute the primary reservoir of these clones and that opportunistic birds like gulls might have been infected from hedgehogs.

The five subclusters which included most of the avian wildlife isolates also contained isolates from several domestic animal species and from feed plant samples. The PFGE patterns of isolates from cats and passerines were identical (A2 and B3), supporting an epidemiological link reported previously in a Swedish study, using phage type analysis (32). Sparrows frequently visit animal sheds and barns in Norway during the winter, and it is thus not surprising that carrier birds could represent a potential health hazard to domestic animals. Previously, we have demonstrated that passerines, including sparrows, maintain a reservoir of serovar Typhimurium O:4,12 (Refsum, Vikøren, et al., submitted). The present results also support the assumption that serovar Typhimurium may be transmitted from gulls to domestic animals or vice versa. Transmission of different salmonella serovars from gulls to cattle and sheep through contaminated drinking water and pastures has previously been suggested (10, 20, 29, 35). Since gulls and pigeons often visit feed and food plants, it is not unreasonable to consider wild birds a potential source of contamination of the factory environment or vice versa.

In Norway, only a few domestically acquired cases of human salmonellosis caused by the multiresistant isolates of serovar Typhimurium DT 104 have been reported (25), and the sources of infection in these cases remain unknown. The two isolates of multiresistant serovar Typhimurium DT 104 reported in this study represent the first known isolates from sources other than humans in Norway. Multiresistant serovar Typhimurium DT 104 has not yet been detected in wild-living birds in Norway. None of the avian isolates in our study exhibited a XbaI PFGE profile identical to the DT 104 prevailing in Europe (4). However, this phage type has previously been detected in passerines, pigeons, and gulls in other countries (7, 18, 19), although information on antibiotic resistance is lacking in these studies.

The L pattern demonstrated in isolates from poultry and swine in our study was also seen in a human outbreak in 1993, but it has not been possible to establish an epidemiological link, and the primary source of infection still remains unknown (Norwegian Institute of Public Health, unpublished data).

Interestingly, we detected a relationship between the PFGE patterns in our study and the phage types previously obtained from analysis of passerine isolates of serovar Typhimurium O:4,12 (27). Characteristic PFGE patterns of different phage types have previously been demonstrated, including serovar Typhimurium DT 104 (4, 5, 7, 16). In this study, PFGE patterns proved to be stable over time, a finding supported by others (5). The patterns of the passerine isolates within the most common subclusters were identical, in spite of a range of 20 to 30 years in their date of collection. This suggests that PFGE is a robust method, not only for discriminative short-term outbreak investigations but also for showing epidemiological significance and applicability in long-term surveillance of serovar Typhimurium epidemiology.

REFERENCES

  • 1.Aavitsland, P., and T. Hofshagen. 1999. Outbreak of salmonellosis in Herøy municipality. Nytt fra miljø- og samfunnsmedisin, vol. 3, report no. 12. National Institute of Public Health, Oslo, Norway. (In Norwegian.)
  • 2.Anderson, E. S., L. R. Ward, M. J. Saxe, and J. D. de Sa. 1977. Bacteriophage-typing designations of Salmonella typhimurium. J. Hyg. 78:297-300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Arbeit, R. 1999. Laboratory procedures for the epidemiologic analysis of microorganisms, p. 116-137. In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken (ed.), Manual of clinical microbiology, 7th ed. American Society for Microbiology, Washington, D.C.
  • 4.Baggesen, D. L., D. Sandvang, and F. M. Aarestrup. 2000. Characterization of Salmonella enterica serovar Typhimurium DT 104 isolated from Denmark and comparison with isolates from Europe and the United States. J. Clin. Microbiol. 38:1581-1586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Bender, J. B., C. W. Hedberg, D. J. Boxrud, J. M. Besser, J. H. Wicklund, K. E. Smith, and M. T. Osterholm. 2001. Use of molecular subtyping in surveillance for Salmonella enterica serotype Typhimurium. N. Engl. J. Med. 344:189-195. [DOI] [PubMed] [Google Scholar]
  • 6.Benton, C., F. Khan, P. Monaghan, W. N. Richards, and C. B. Shedden. 1983. The contamination of a major water supply by gulls (Larus sp.). Water Res. 17:789-798. [Google Scholar]
  • 7.Besser, T. E., C. C. Gay, J. M. Gay, D. D. Hancock, D. Rice, L. C. Pritchett, and E. D. Erickson. 1997. Salmonellosis associated with S. typhimurium DT 104 in the USA. Vet. Rec. 140:75. [PubMed] [Google Scholar]
  • 8.Callow, B. R. 1959. A new phage-typing scheme for Salmonella typhi-murium. J. Hyg. 57:346-359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Clark, M. R., and D. E. Woodward. 1992. Generating random numbers with base SAS software. Observations. Tech. J. SAS Software Users 1:12-19. [Google Scholar]
  • 10.Coulson, J. C., J. Butterfield, and C. Thomas. 1983. The herring gull Larus argentatus as a likely transmitting agent of Salmonella montevideo to sheep and cattle. J. Hyg. 91:437-443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Fenlon, D. R. 1981. Seagulls (Larus spp.) as vectors of salmonellae: an investigation into the range of serotypes and numbers of salmonellae in gull faeces. J. Hyg. 86:195-202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Fennel, H., D. B. James, and J. Morris. 1974. Pollution of a storage reservoir by roosting gulls. Water Treat. Exam. 23:5-24. [Google Scholar]
  • 13.Girdwood, R. W. A., C. R. Fricker, D. Munro, C. B. Shedden, and P. Monaghan. 1985. The incidence and significance of salmonella carriage by gulls (Larus spp.) in Scotland. J. Hyg. 95:229-241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Glünder, G. 1989. Infections of pigeons as a risk to the health of humans and animals. Dtsch. Tierärztl. Wochenschr. 96:112-116. (In German.) [PubMed] [Google Scholar]
  • 15.Handeland, K., G. Kapperud, T. Refsum, B. Strøm Johansen, G. Holstad, G. Knutsen, I. Solberg, and J. Schultze. 2002. Prevalence of Salmonella Typhimurium in Norwegian hedgehog populations associated with two human disease outbreaks. Epidemiol. Infect. 128:523-527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Heir, E., B.-A. Lindstedt, I. Nygård, T. Vardund, V. Hasseltvedt, and G. Kapperud. 2002. Molecular epidemiology of Salmonella Typhimurium isolates from human sporadic and outbreaks cases. Epidemiol. Infect. 128:373-382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Heir, E., B.-A. Lindstedt, T. Vardund, Y. Wasteson, and G. Kapperud. 2000. Genomic fingerprinting of shigatoxin-producing Escherichia coli (STEC) strains: comparison of pulsed-field gel electrophoresis (PFGE) and fluorescent amplified-fragment-length polymorphism (FAFLP). Epidemiol. Infect. 125:537-548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Hubálek, Z., W. Sixl, M. Mikulášková, V. B. Sixl, W. Thiel, J. Halouzka, Z. Juřicova, B. Rosicky, L. Mátlová, M. Honza, V. Hájek, and J. Sitko. 1995. Salmonellae in gulls and other free-living birds in the Czech Republic. Centr. Eur. J. Public Health 3:21-24. [PubMed] [Google Scholar]
  • 19.Humphrey, T. 2001. Salmonella Typhimurium definitive type 104. A multi-resistant salmonella. Int. J. Food Microbiol. 67:173-186. [DOI] [PubMed] [Google Scholar]
  • 20.Johnston, W. S., G. K. Maclachlan, and G. F. Hopkins. 1979. The possible involvement of seagulls (Larus sp) in the transmission of salmonella in dairy cattle. Vet. Rec. 105:526-527. [PubMed] [Google Scholar]
  • 21.Kapperud, G., S. Gustavsen, I. Hellesnes, A. H. Hansen, J. Lassen, J. Hirn, M. Jahkola, M. A. Montenegro, and R. Helmuth. 1990. Outbreak of Salmonella typhimurium infection traced to contaminated chocolate and caused by a strain lacking the 60-megadalton virulence plasmid. J. Clin. Microbiol. 28:2597-2601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Kapperud, G., J. Lassen, K. Dommarsnes, B. E. Kristiansen, D. A. Caugant, E. Ask, and M. Jahkola. 1989. Comparison of epidemiological marker methods for identification of Salmonella typhimurium isolates from an outbreak caused by contaminated chocolate. J. Clin. Microbiol. 27:2019-2024. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Kapperud, G., H. Stenwig, and J. Lassen. 1998. Epidemiology of Salmonella typhimurium O:4-12 infection in Norway. Evidence of transmission from an avian wildlife reservoir. Am. J. Epidemiol. 47:774-782. [DOI] [PubMed] [Google Scholar]
  • 24.Leegaard, T. M., D. A. Caugnat, L. O. Froholm, E. A. Høiby, and J. Lassen. 2000. Emerging antibiotic resistance in Salmonella Typhimurium in Norway. Epidemiol. Infect. 125:473-480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Norwegian Institute of Public Health. 1997-2001. The Norwegian Surveillance System for Communicable Diseases, MSIS. Norwegian Institute of Public Health, Oslo.
  • 26.On, S. L., and D. L. Baggesen. 1997. Determination of clonal relationships of Salmonella typhimurium by numerical analysis of macrorestriction profiles. J. Appl. Microbiol. 83:699-706. [DOI] [PubMed] [Google Scholar]
  • 27.Refsum, T., K. Handeland, D. L. Baggesen, G. Holstad, and G. Kapperud. 2002. Salmonellae in avian wildlife in Norway from 1969 to 2000. Appl. Environ. Microbiol. 68:5595-5599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Scholtens, R. T., and G. Caroli. 1971. Role of pigeons in the spread of salmonellosis: incidence of different types of Salmonella typhi-murium var. copenhagen in pigeons, man, and other animals. Antonie Leeuwenhoek 37:473-476. [DOI] [PubMed] [Google Scholar]
  • 29.Sharp, J. C. M., W. J. Reilly, K. A. Linklater, D. M. Inglis, W. S. Johnston, and J. K. Miller. 1983. Salmonella montevideo infection in sheep and cattle in Scotland, 1970-81. J. Hyg. 90:225-232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Søbstad, Ø., J. Blinkenberg, E. Bergesen, A. Digranes, I. Tveit, E. Heir, G. Kapperud, T. Stavnes, V. Hasseltvedt, and B. G. Iversen. 2000. Transmission of salmonellosis through hedgehogs in Norway. Eurosurveillance Weekly, vol. 4, no. 38.
  • 31.Tamada, Y., Y. Nakaoka, K. Nishimori, A. Doi, T. Kumaki, N. Uemura, K. Tanaka, S. I. Makino, T. Sameshima, M. Akiba, M. Nakazawa, and I. Uchida. 2001. Molecular typing and epidemiological study of Salmonella enterica serotype Typhimurium isolates from cattle by fluorescent amplified- fragment length polymorphism fingerprinting and pulsed-field gel electrophoresis. J. Clin. Microbiol. 39:1057-1066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Tauni, M. A., and A. Österlund. 2000. Outbreak of Salmonella typhimurium in cats and humans associated with infection in wild birds. J. Small Anim. Pract. 41:339-341. [DOI] [PubMed] [Google Scholar]
  • 33.Van Oye, E., and J. Borghijs. 1979. Do pigeons play a role in human infections with Salmonella typhimurium var. copenhagen? Dtsch. Tierärztl. Wochenschr. 86:306-307. (In German.) [PubMed] [Google Scholar]
  • 34.Vidar, G., and M. Haugum. 1997. Outbreak of salmonellosis in Moss during the autumn, 1996. Norsk Veterinærtidsskr. 109:33-35. (In Norwegian.) [Google Scholar]
  • 35.Williams, B. M., D. W. Richards, D. P. Stephens, and T. Griffiths. 1977. The transmission of S. livingstone to cattle by the herring gull (Larus argentatus). Vet. Rec. 100:450-451. [DOI] [PubMed] [Google Scholar]
  • 36.Wuthe, H. H. 1971. Studies on types of Salmonella typhimurium in domestic pigeons in Schleswig-Holstein and their possible epizootological significance. Berl. Münch. Tierärztl. Wochenschr. 84:290-292. (In German.) [PubMed] [Google Scholar]
  • 37.Wuthe, H. H. 1972. Importance of the domestic pigeon in human salmonellosis. Dtsch. Tierärztl. Wochenschr. 79:507-508. (In German.) [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES