Molecular Follow-Up of Salmonella enterica subsp. enterica Serovar Agona Infection in Cattle and Humans

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Oct. 2002, p Vol. 40, No /02/$ DOI: /JCM Copyright 2002, American Society for Microbiology. All Rights Reserved. Molecular Follow-Up of Salmonella enterica subsp. enterica Serovar Agona Infection in Cattle and Humans Nanna Lindqvist, 1 Anja Siitonen, 2 and Sinikka Pelkonen 1 * National Veterinary and Food Research Institute, Kuopio Department, FIN Kuopio, 1 and National Public Health Institute, Laboratory of Enteric Pathogens, FIN Helsinki, 2 Finland Received 23 January 2002/Returned for modification 11 April 2002/Accepted 2 July 2002 Salmonella enterica subsp. enterica serovar Agona was not frequently encountered in Finland until an increase in rates of isolation among animal and feed was seen in A small outbreak among cattle farms in the regions of Oulu and Vaasa in northwestern Finland in included eight farms. After the outbreak, an increase in the number of serovar Agona infections in humans was seen in 1999: the number of annual microbiologically confirmed cases in humans increased from about 10 from 1990 to 1998 to 84 in 1999, including an outbreak in which more than 50 people were infected. To gather epidemiological data on serovar Agona and to trace the origin of the human infections, 110 serovar Agona isolates isolated from animal, feed, and other sources as well as from humans with cases of salmonellosis of domestic and foreign origin, which were recovered from 1984 to 1999, were analyzed for their pulsed-field gel electrophoresis (PFGE), plasmid, and IS200 profiles and antibiograms. Of these typing methods, PFGE with restriction endonucleases XbaI, BlnI, NotI, and SpeI was the most useful. The PFGE profile of the strain causing an outbreak among cattle in Finland in was not seen previously. The strain with this profile was later only sporadically found in human infections. The profile of the strain causing the human outbreak in 1999 was not found among isolates from cattle or any other sources. Molecular typing was valuable in showing that although the outbreaks in cattle and humans seemed to be related regionally, they were not related otherwise. Salmonella enterica subsp. enterica serovar Agona (antigenic structure, 4,12:fgs: ) was first isolated from cattle in Ghana (10). In 1969 and 1970, serovar Agona emerged as a public health problem in the United States, the United Kingdom, The Netherlands, and Israel. Its appearance was attributed to imported Peruvian fish meal. Subsequently, serovar Agona was recovered from domestic animals and also from humans (6). Its persistence in livestock has probably resulted from the recycling of treated animal wastes as feed (23). Bartolozzi et al. (2) reported on an outbreak of serovar Agona in a ward for infants in an Italian hospital in Recently reported large serovar Agona outbreaks have been associated with a ready-to-eat savory snack (11, 19) and with a toasted-oats cereal (5). Serovar Agona has not been common in Finland. Of the serovar Agona strains isolated from humans, 69 to 100% have been from Finnish tourists who had been abroad preceding their salmonella infection (16). Only sporadic serovar Agona isolations (less than three annually) had been made among animals before 1994 (17). At the end of 1994, serovar Agona was isolated from cattle at two farms. Serovar Agona-infected cattle were detected at six additional farms in The farms involved in this outbreak were located in six governmental districts, four near the River Kalajoki in the province of Oulu (six farms) and two geographically nearby in the province of Vaasa (one farm each). The source for the outbreak was not found, and the time required for the elimination of the infection varied between less than 2 months (three farms) and more * Corresponding author. Mailing address: National Veterinary and Food Research Institute, Kuopio Department, P.O. Box 92, FIN Kuopio, Finland. Phone: Fax: sinikka.pelkonen@eela.fi. than 12 months (one farm) (1). Serovar Agona was the third most common salmonella serovar infecting cattle in Finland in 1995, after serovars Infantis and Typhimurium (17). This study was undertaken to gather data on the molecular epidemiology of serovar Agona infections in animals and humans and to find out whether an increase in the number of animals found to be infected with this serovar has any effect on the number of cases of infection in humans caused by serovar Agona. During the study, a large human outbreak involving more than 50 cases of serovar Agona infection occurred in November and December 1999 (Fig. 1). The domestic outbreak was related to guests at the restaurant of a spa hotel in the region of Vaasa. Epidemiological investigations were carried out by local authorities, but the source of the outbreak remained unknown. MATERIALS AND METHODS Serovar Agona isolates. The isolates (total, n 110) were obtained from the laboratories of the National Veterinary and Food Research Institute of Finland (n 62); the National Public Health Institute (KTL), Helsinki, Finland (n 41); the Plant Production Inspection Centre, Vantaa, Finland (n 2); the laboratory of the Fur Animal Association, Vaasa, Finland (n 2); and two commercial feed-producing companies in Finland (n 3). The strains were isolated from cattle, animal feed, fur animals, humans, and other sources (Table 1) from 1984 to The first outbreak of serovar Agona infection among cattle in Finland occurred in and involved eight farms. Isolates collected from these affected farms as well as other cattle isolates (n 32) isolated from 1984 to 1999 and isolates (n 28) from imported products, feed, fur animals and other animals, and sewage water were analyzed. The isolates from humans were classified by the Laboratory of Enteric Pathogens of KTL as either domestic (a patient had not been abroad during the month preceding the date when a specimen was taken) or foreign. Thirty of the human isolates had been associated with recent foreign travel. Isolates from sporadic cases of domestic origin from 1996 and 1997 and three isolates from an outbreak in 1999 involving over 50 people were also analyzed. All isolates had been serologically confirmed to be serovar Agona and stored at 70 C. 3648

2 VOL. 40, 2002 MOLECULAR TYPING OF S. ENTERICA SEROVAR AGONA 3649 FIG. 1. Occurrence of domestic (E) and imported (}) human cases of Salmonella serovar Agona infection from 1999 through Yr Cattle farm PFGE. Chromosomal DNA was prepared in gel blocks, and the agarose plugs were treated as described previously (13). Restriction enzyme and S1 nuclease analyses (see below) were performed with slices from the same agarose plugs. The slices were digested at 37 C for 16 to 18 h with either 20 U of XbaI ornoti or 12 U of SpeI in the reaction buffer supplied by the manufacturer (New England Biolabs, Beverly, Mass.). Digestion with 5 U of BlnI was done at 37 C for 4 h in the reaction buffer supplied by the manufacturer (Boehringer Mannheim GmbH, Mannheim, Germany). All 110 strains were digested with XbaI, and strains representing each pulsed-field gel electrophoresis (PFGE) type as detected by XbaI restriction were analyzed with other enzymes (52 strains altogether). PFGE was performed as described previously (13). Briefly, for the XbaI-treated plugs the electrophoretic conditions were electrophoresis for 19 h at 14 C with a pulse ramp time of 10 to 30 s, a voltage of 6 V/cm, and a reorientation angle of 120. The pulse ramp time was changed to 1 to 20 s for the NotI- and SpeI-digested plugs and to 10 to 40 s for the BlnI-digested plugs. The molecular weights of the linearized plasmids (see below) and restriction fragments were determined by plotting the distance of migration against the log 10 of the fragments (18, 20) of the bacteriophage molecular size ladder. DNA profiles differing by one or more DNA fragments were assigned a pulsed-field (pf) type number (XbaI restriction) or letter (other enzymes). The coefficient of similarity (F) values between the pf types were calculated as described previously (7). After visual analysis of the PFGE profiles, a computer program for analysis of electrophoretic patterns (GelCompar; Applied Maths, Kortrijk, Belgium) (24) was used to generate dendrograms. Plasmid analyses. Plasmids smaller than 20 kb were isolated by the alkaline lysis method described by Grinsted and Bennett (9). The preparations were analyzed in 0.9% agarose gels (SeaKem LE; FMC Bioproducts, Rockland, Maine) at 4 V/cm for 1.5 h in 1 Tris-acetate-EDTA buffer (18), and the gels were stained with 0.5 g of ethidium bromide per ml. Plasmids larger than 20 kb were analyzed by PFGE. The agarose plugs used for PFGE were treated with S1 nuclease, which linearizes plasmids (3), thereby making plasmid analysis and their size determination easier. S1 nuclease treatment was done at 37 C for 45 min in the reaction buffer supplied with the enzyme (code M576/1,2; Promega, Madison, Wis.). The analyses were performed with slices from the same agarose plugs used for chromosomal profiling by PFGE (see above). Escherichia coli strains V517 (plasmids of 35.6, 4.8, 3.7, 3.4, 1.8, and 1.4 MDa) (14) and 39R861 (plasmids of 98.0, 42.0, 23.9, and 4.6 MDa) (21) were used as plasmid reference strains in all alkaline lysis isolation procedures. PCR for detection of IS200. The primer pair 5 -CCTAACAGGCGCATACG ATC-3 and 5 -ACATCTTGCGGTCTGGCAAC-3 (4) and a 30-cycle program (1 min at 94 C, 0.5 min at 54 C, and 2 min at 72 C) were used to amplify a 557-bp PCR product of the IS200 insertion sequence. One of our own isolates of serovar Infantis (strain K1469) that was positive for IS200 was used as a control in all PCR runs. Testing for microbial drug resistance. The agar diffusion test was performed as described by NCCLS with Oxoid (Basingstoke, United Kingdom) disks and Mueller-Hinton agar (Becton Dickinson and Company, Cockeysville, Md.). The disks contained 10 g of ampicillin, 30 g of cefotaxime, 30 g of chloramphenicol, 5 g of ciprofloxacin, 5 g of enrofloxacin, 10 g of streptomycin, 25 g of sulfamethoxazole-trimethoprim, and 30 g of tetracycline. RESULTS Profiles obtained by PFGE with XbaI. Digestion of all 110 strains with XbaI yielded fragments in the size range of 40 to 600 kb and 39 different pf types (Table 2; Fig. 2). Nineteen and 23 pf types were seen among the 69 nonhuman isolates and the 41 human isolates, respectively (Table 2). Of the 39 pf types, 31 were seen in only one or two isolates each (Table 2). The most common profile was pf1 (22 strains), followed by pf21 (13 strains), pf22 (9 strains), pf5 (8 strains), and pf19 (7 strains). Strains of these five types represented 54% (59 strains) of the 110 strains studied. Of these, pf5 was seen only among human strains, with the other most common types seen only among nonhuman strains (excluding one human domestic strain of pf1). Of the 39 pf types, 14 were seen in isolates of foreign TABLE 1. Origin and number of Salmonella serovar Agona isolates analyzed Cattle slaughterhouse or transport vehicle No. of isolates with the following origin: domestic foreign a Fur animals or their feed b Feed c Other d Total e Total a Human isolates with recent history of foreign travel. The isolates were from 15 different countries in four geographical areas: Asia, n 19; Europe, n 6; the Americas, n 3; Africa, n 2. b Fur animal feed (including slaughterhouse by-products). c Domestic feed, n 8; imported feed, n 3. d For 1984 to 1987, imported food, a dog, and sewage water; for 1994, imported food; for 1996, foreign pig isolate; for 1999, a badger and imported fertilizer made of horn. e From an epidemic.

3 3650 LINDQVIST ET AL. J. CLIN. MICROBIOL. TABLE 2. Distribution of the 39 XbaI profiles (pf1 to pf40) among 110 Salmonella serovar Agona isolates with respect to origin and year of isolation Profile for isolates with the following origins a : Yr Cattle farm Cattle slaughterhouse or transport vehicle b domestic foreign Fur animals and their feed Feed Other c 1984 pf pf pf12, pf pf pf13, pf pf1 d pf pf1 d, pf2 d, pf3 pf4 (A) pf5, pf6, pf7, pf8, pf11, pf16, pf17 pf1 d 1996 pf1 d (B) pf16, pf24, pf29 pf7, pf24, pf26, pf28, pf29, pf31, pf32, pf34, pf36, pf37 pf1 d pf pf21, pf22 pf1 d (C), pf21, pf22, pf23 (D) pf1, d pf5, pf27, pf33 pf5, pf25, pf27, pf30, pf35, pf pf1 d (E) pf39 e pf38 pf19 pf40 a Only the pf types that differ from each other are shown. b Five different slaughterhouses, indicated A to E. c For 1984, imported food; for 1985, a dog; for 1987, sewage water; for 1994, imported food; for 1996, foreign pig isolate; for 1999, a badger and imported fertilizer made of horn. d Profile for isolates from the outbreak among cattle. e Profile for isolates from the outbreak among humans. origin only, 5 were seen in human strains of foreign and domestic origin, and 3 were seen in human strains of domestic origin only. The overall differences between pf1 and the 38 other pf types obtained by XbaI digestion (pf1 to pf40) gave Dice similarity coefficient values larger than 0.6 (Fig. 2). XbaI profiles pf1 and pf2 were associated with the outbreak among cattle farms in 1994 and 1995, and profile pf39 was associated with the outbreak among humans in Only one isolate from humans had the profile for isolates from the cattle outbreak and none of the cattle isolates belonged to pf39. pf1 and pf2 differed from each other only by an intensive band of approximately 100 kb, and S1 nuclease analysis showed that pf2 contained a plasmid of that size (Table 3). pf39 differed from pf1 by nine bands (Fig. 2). PFGE profiles obtained by digestion with BlnI, NotI, and SpeI. Since the overall differences between the XbaI profiles were not large (Fig. 2), one isolate of each 39 XbaI pf type (Table 2) and additional isolates of the most common XbaI pf type, pf1, were analyzed with other enzymes as well (Table 3). Among the 52 isolates analyzed, digestion with BlnI yielded fragments of 40 to 600 kb and 28 different types (designated a to w), and digestion with NotI yielded fragments of 10 to 250 kb and 21 types (designated N1 to N21). Digestion with SpeI yielded fragments of 10 to 400 kb and 21 different types (designated A to T) among the 45 isolates analyzed. Although the number of profiles obtained by digestion with any of these enzymes was not as large as the number obtained by digestion with XbaI, most of the BlnI, NotI, and SpeI profiles were seen in only one or two of the isolates analyzed (Table 3). However, the XbaI profiles seen in cattle isolates from 1994, 1995, and 1997 (profile pf1, pf2, pf3, and pf4 and profile pf21, pf22, and pf23) shared SpeI profiles A and J, respectively (Table 3). The differences between these two SpeI profiles, as well as between the BlnI and NotI profiles (Table 3), seen in these cattle isolates are minor (a one- to four-band difference) (data not shown). As for the profiles seen in the isolates representative of the cattle outbreak (XbaI profiles pf1 and pf2) and the human outbreak (pf39) (Table 3), BlnI profile a1 resembled BlnI profile a but had an additional band (data not shown). There was also a one-band difference between SpeI profiles A and AD and NotI profiles N1 and N7 (data not shown). Plasmid profiles. S1 nuclease analysis combined with PFGE showed that 35% (38 of 110) of the strains harbored plasmids larger than 20 kb (data for representative strains are shown in Table 3). Smaller plasmids with six different profiles (profiles a to f) were detected in 15% (16 of 110) of the isolates (Table 3). Possession of IS200. The 557-bp product of IS200 was not amplified from any of the serovar Agona isolates. A strong and clear band of that size was always amplified from control strain K1469. Antimicrobial resistance patterns. Of the 73 domestic isolates, only 1 isolate from a cattle farm (strain 2476) recovered in 1997 showed resistance to any of the antimicrobials tested (chloramphenicol and tetracycline; Table 3). Five of the 37 foreign isolates were resistant, and resistance to tetracycline and streptomycin was the most common (Table 3). Combination of profiles. When representative isolates of each of the 39 XbaI profiles were analyzed with additional restriction enzymes (Table 3), the relatively large number of different profiles obtained with the additional enzymes (28, 21, and 21 profiles for BlnI, NotI, and SpeI, respectively) supports the existence of at least minor differences between the 39 XbaI profiles. The isolate representative of the human outbreak (XbaI profile pf39) did not seem to be related to any of the other isolates tested, whereas two groups (groups A and J) were formed among the cattle isolates by SpeI digestion. Large plasmids were seen in isolates comprising one-third (13 of 39) of the XbaI profiles and small plasmids were seen in isolates comprising one-fifth (8 of 39) of the XbaI profiles, but there seemed to be no connection between the PFGE profiles and plasmid profiles. All drug-resistant strains seemed to harbor large plasmids, thereby indicating the possibility of plasmidmediated drug resistance in the isolates analyzed. Due to the large number of profiles obtained with all restriction enzymes applied in the study and the lack of an obvious connection

4 VOL. 40, 2002 MOLECULAR TYPING OF S. ENTERICA SEROVAR AGONA 3651 Downloaded from FIG. 2. Dendrogram showing 39 pf profiles of Salmonella serovar Agona after XbaI restriction of chromosomal DNA followed by PFGE. Dice similarity coefficients are shown at the upper left; the molecular sizes (in kilobases) of the fragments are shown at the upper middle. XbaI profiles pf1 to pf40 and the number of isolates with the XbaI profile, as well as the human and cattle outbreak profiles, are indicated on the right. between the PFGE and plasmid profiles, the material could not be divided into a few clear combination profile groups. DISCUSSION Salmonella infection in cattle can be transmitted to humans by several routes, some of which, such as the environment, are difficult to control. The endemic serovars Infantis and Typhimurium persist among cattle in Finland, and although the prevalence rate of these serovars is less than 2% for the farms affected, these serovars play a major role in human salmonelloses of domestic origin. Therefore, the first notification of the Agona outbreak in cattle in made us alert, and we started to survey the Agona strains by molecular typing. The purpose was to see how the strains causing infections evolve and spread and what impact they have on human health. Such scientific data are required for, for example, assessment of risk for Salmonella infections. Among the typing methods applied in this study, PFGE proved to be the most useful. The serovar Agona isolates did not harbor any IS200 element, as has also been reported previously (8, 12), making this method inapplicable. It is also unlikely that serovar Agona can be subdivided by ribotyping (22). With few exceptions, the isolates were sensitive to all antimicrobials tested; thus, antibiograms did not provide useful epidemiological markers. Threlfall et al. (22) described the application of PFGE with XbaI digestion to an international outbreak strain of serovar Agona. Different PFGE conditions prevent direct comparison of their results and ours, but serovar Agona seems to have many conserved restriction sites for the XbaI restriction enzyme, since many fragments are common between our different on October 31, 2018 by guest

5 3652 LINDQVIST ET AL. J. CLIN. MICROBIOL. TABLE 3. Combined results after XbaI, BlnI, SpeI, and NotI restriction, plasmid analyses, and susceptibility testing for 39 representative isolates of Salmonella serovar Agona Strain Yr Origin Profile by PFGE with: XbaI BlnI SpeI NotI PFGE profiles. Double bands, i.e., bands containing two fragments of roughly the same size but visible as one thick band, are commonly produced from serovar Agona by digestion with XbaI. The presence of plasmids larger than 20 kb complicates the interpretation of banding patterns when the fragments generated are small, e.g., after digestion with NotI and SpeI, in which the fragment sizes are approximately 10 kb and higher. We found a total of 39 different PFGE types by XbaI digestion of the 110 strains analyzed. The overall differences between the types were small, and most types were encountered in only one or two isolates. The importance of the ability to differentiate the isolates into that many subgroups can be questioned, and too many conclusions should perhaps not be drawn on the basis of the results obtained with only one restriction enzyme. Analysis with additional enzymes (BlnI, SpeI, and NotI), however, supported our findings that although the pf types by XbaI were highly similar, the differences seen cannot be disregarded. The situation was the opposite only for pf types Size(s) (kb) of large plasmids Plasmid analysis Profile for small plasmids 1301 b 1995 Cattle pf1 a A N b 1995 Cattle pf2 a A N b 1995 Cattle pf3 b A N Cattle pf4 f A N1 IHS Human pf5 e C N2 a IHS Human pf6 e L N2 IHS Human pf7 n D N Other pf8 c C N20 a E Other pf9 g M N a C, S, TE Other pf10 c E N Other pf11 h C N Cattle pf12 i F N5 95 b Other pf13 j G N6 E Other pf14 j G N Cattle pf15 k J N6 IHS Human pf16 d H N8 IHS Human pf17 l B N Other pf19 a C N12 c Other pf20 m K N21 126, 106, 92 d C, S, SXT Cattle pf21 d J N13 82 C, TE Cattle pf22 o J N Cattle pf23 o J N IHS Human pf24 c M N2 IHS Human pf25 p N N10 IHS Human pf26 q O N2 66 C, S, TE IHS Human pf27 r C N2 IHS Human pf28 j P N7 IHS Human pf29 s DH N14 IHS Human pf30 t Q N15 120, 27 TE IHS Human pf31 u R N16 44 S, TE IHS Human pf32 v S N17 91, 41 e IHS Human pf33 l B N11 IHS Human pf34 c M N2 87 IHS Human pf35 x L N2 f IHS Human pf36 y J N7 IHS Human pf37 z M N18 IHS Human pf38 w C N15 IHS c 1999 Human pf39 a1 AD N Badger pf40 e1 T N2 a R, drug resistance profile. C, chloramphenicol; S, streptomycin; SXT, sulfamethoxazole-trimethoprim; TE, tetracycline. b Representative of isolates from outbreak among cattle. c Representative of isolates from outbreak among humans. pf1, pf2, pf3, pf4, pf21, pf22, and pf23, in which analysis with other enzymes in addition to XbaI supported our conclusions that these pf types are closely related. Four XbaI profiles (profiles pf1, pf2, pf3, and pf4) were associated with serovar Agona infections on cattle farms in These profiles were not previously seen among domestic or foreign isolates. pf1 was seen in the same area of northwestern Finland in slaughterhouse hygiene samples in 1996 (slaughterhouse B) and 1997 (slaughterhouse C) and in fur animals in Fur animals are often fed slaughterhouse waste, and although it is usually treated with heat or formic acid to avoid contamination, Salmonella is sometimes isolated from fur animals. Only one domestic human isolate of pf1 was detected, and it was in 1997, long after the outbreak in cattle. Three XbaI profiles (profiles pf21, pf22, and pf23) seemed to be related to a small outbreak among cattle in Finland in The difference of three to six bands between these pf types and pf1 or pf2 can be explained by point mutations and suggests R a

6 VOL. 40, 2002 MOLECULAR TYPING OF S. ENTERICA SEROVAR AGONA 3653 that the outbreaks in and 1997 were possibly caused by related strains (15, 20). These three pf types (pf21, pf22, and pf23) share SpeI profile J. pf22 and pf23 also share BlnI profile o and NotI profile N14. Compared to XbaI profile pf21, the two additional bands (200 and 130 kb) of pf23 may have resulted from a point mutation. The molecular sizes of the additional bands add up to the molecular size of the missing band (330 kb) of pf23. All of these three pf types harbored one large plasmid, the size of which varied from 82 to 100 kb. The outbreak among humans in 1999 occurred in the region of Vaasa, which is relatively close to the cattle farm outbreak in There seemed to be a connection with fur animals, which were fed waste picked up from the restaurant associated with the human outbreak. One would therefore have thought it likely that profile pf1 or pf2 instead of a new profile, profile pf39, would have been detected in the human outbreak isolates. However, there is at least a seven-band difference between profiles pf1 and pf39, thereby indicating that the profiles are unrelated (15). pf1 was still seen in slaughterhouse isolates (slaughterhouse E) from 1999 in southeastern Finland, but on the basis of molecular typing, it is much more likely that the human outbreak in 1999 was not related to traces of the cattle farm outbreak in Without detailed typing results, an infection route from cattle (1994) to fur animals (1996) and, finally, to humans (1999) would have easily been accepted as the cause of the human epidemic. None of the isolates of foreign origin had XbaI profile pf1, pf2, pf3, pf4, pf21, pf22, or pf23, which were seen in domestic cattle isolates, nor did they have profile pf39, which was related to the restaurant-associated outbreak among humans in However, no recent foreign isolates were available, so a foreign source for the restaurant-associated outbreak that affected humans cannot be disregarded. The many XbaI profiles seen among the 30 human isolates of foreign origin (21 profiles, 14 of which were seen only among the foreign isolates) may reflect the heterogeneity of the worldwide serovar Agona infection. Five profiles (profiles pf5, pf16, pf24, pf27, and pf29) were also seen among human isolates of domestic origin. It cannot be excluded that some isolates were misclassified as foreign or that some infections classified as domestic in fact have a foreign origin. In 1996, 1997, 1998, and 1999, four, seven, two, and one cattle farms, respectively, were serovar Agona positive (17). On the basis of the typing results for isolates recovered after those involved in the outbreak in , we suggest that a new strain invaded the area and caused the infections in cattle in 1994 and that, although we do not know where the original outbreak strain with XbaI profile pf1 or pf2 came from, it is highly likely that a closely related strain caused the infection among cattle in The outbreak among cattle in was not associated with the outbreak among humans in 1999, nor does it seem to have affected the annual level of domestic serovar Agona infections among humans in Finland (16). The ability to make these conclusions shows the importance of knowing the molecular epidemiology of Salmonella infections occurring today. ACKNOWLEDGMENTS This work was supported by grants MMM 4548/507/94 and MMM 5402/501/96 from the Ministry of Agriculture and Forestry of Finland. 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