FEMS Microbiology Letters 187 (2000) 21^25

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1 FEMS Microbiology Letters 187 (2000) 21^25 Persistence of a Salmonella enterica serotype Typhimurium clone in Danish pig production units and farmhouse environment studied by pulsed eld gel electrophoresis (PFGE) Dorthe Sandvang *, Lars B. Jensen, Dorte Lau Baggesen, Suraj B. Baloda Danish Veterinary Laboratory, Bu«lowsvej 27, DK-1790 Copenhagen V, Denmark Received 17 February 2000; received in revised form 31 March 2000; accepted 4 April 2000 Abstract The clonal relationship among Salmonella enterica serotype Typhimurium isolates from selected pig production units in Denmark was investigated by the pulsed field gel electrophoresis (PFGE) typing method to determine environmental survival and spread of Salmonella in different herds. Thirty-four Typhimurium isolated during 1996^1998 from porcine faeces and environmental samples from three pig farms designated 1, 3 and 5 were characterised by PFGE using two restriction enzymes. Farm 5 supplied piglets to farm 1 and the herds were located close to each other. Results of PFGE analysis showed both intra- and inter-relationships, i.e. identical PFGE patterns among the faecal and environmental isolates from farm 1 and farm 5. All the isolates from farm 3 irrespective of the source showed identical PFGE patterns, but were different from samples from farms 1 and 5. This study indicates spread between farms and survival of a farm-specific clone. Furthermore, identical PFGE patterns of isolates from piglet supplier and finisher herds indicate that the farrow-to-grower herd of farm 5 was sub-clinically infected prior to delivery to farm 1 and thereby caused the transmission of Salmonella. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Salmonella enterica Typhimurium; Pulsed eld gel electrophoresis typing; Persistence; Agriculture; Farm 1. Introduction Salmonella is still one of the major zoonotic pathogens worldwide and despite the use of extensive prevention strategies and attempts to reduce the infection in animals and humans, it still remains a serious problem [1]. Outbreak of food-borne disease associated with pig products is a human health problem and also has economic importance in Denmark where the annual production is about 23 million pigs [2]. An investigation in the USA showed that approximately one quarter of the porcine faecal samples were Salmonella-positive, of which S. enterica serovar Typhimurium was one of the predominant serotypes [3]. In Denmark 3% of the individual pigs brought to the slaughterhouse were Salmonella-positive in 1998 representing a 50% decrease compared to the ndings in 1993 and * Corresponding author. Present address: Statens Serum Institute, 5 Artillerivej, DK-2300 Copenhagen S, Denmark. Tel.: ; Fax: ; ds@ssi.dk 1994 [4,5]. A wide variety of phage types and genotypes of Typhimurium have previously been identi ed in Danish pig production [6]. The most frequently isolated phage type of Typhimurium in 1993/1994 as well as in 1998 was de nitive phage type DT12 [4,7]. Previous investigations have shown that this phage type mainly has been spread clonally by trading of animals [8]. On the other hand, Mutalib et al. [9] have reported the isolation of identical phage types of S. enterica serovar Enteritidis in clinical and in environmental samples from poultry, indicating clonal survival. Similarly, the persistence of Enteritidis in poultry and Typhimurium in cattle herd environments has been described by other authors, suggesting recrudescence of a single clone [10,11]. McLaren and Wray [12] described the persistence of Typhimurium DT204c in cattle farms where one farm harboured a DT204c strain with a speci c plasmid pro le that was detected over a period of 19 months. In Denmark the same genotype of multi-resistant Typhimurium DT104 was repeatedly isolated from several herds for up to 16 months [7] and, in a single case, identical isolates were recovered after 6 years (Baggesen, unpublished ob / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (00)

2 22 D. Sandvang et al. / FEMS Microbiology Letters 187 (2000) 21^25 servations). However, it is not clear whether persistence is due to chronic sub-clinical infection in pigs or the persistence of Salmonella in the environment. Thus, the present study was directed towards the study of persistence of Salmonella in the environment and the probable routes of transmission. To study the persistence of Typhimurium in selected individual pig-producing units, we genotyped isolates from the environment and from sub-clinically infected animals of three selected pig farms in Denmark. 2. Materials and methods 2.1. Pig farms Three Danish pig farms selected for this study were designated farm Nos. 1, 3 and 5. Farms 1 and 3 were part of the larger animal husbandry and environment investigations being carried out on local pigs farms (designated farms 1, 2, 3 and 4) at the Danish Veterinary Laboratory. All farms had a history of recurrent clinical and sub-clinical Salmonella infections. Farm 5 supplied piglets to farm 1 while farm 3 was a farrow-to- nisher unit and sold the pigs directly to the slaughterhouse. These farms disposed of the farmhouse waste on their agricultural elds once a year. Samples of farm manure, slurry and soil from their agricultural elds were collected and investigated for the presence of Salmonella. Animal waste from farm 1 was solid, straw-based and disposed of on a manure dump on the farm premises. Farm 1 used a conveyor belt to transport manure from the piggery to the dump. Farm 3 waste from piggeries consisted of slurry collected and stored in large slurry tanks. The environmental conditions of farm 5 were not known or investigated since the isolate included in this investigation was a clinical sample submitted to our laboratory for suspected Salmonella infection Bacterial isolates Samples from animals and environment were tested for the presence of Salmonella by pre-culture in enriched bu - ered peptone water and use of selective media [4]. Samples for investigations were collected from: (a) untreated soil (soil on which no slurry or manure had been spread for at least a year), (b) treated soil (slurry or manure spread on the agricultural land during the past year or annually), (c) piggeries, (d) manure conveyor belt, (e) manure dump outer surface, (f) manure dump inner region, from a depth of approximately 20 cm, (g) slurry, (h) feed, and (i) porcine faecal samples. The soil samples were taken by removing an upper 10-cm layer from the surface. In the case of `treated soil', the samples were collected a short while after the manure/slurry had been spread, and later when the soil was ploughed to mix and distribute the fertiliser. It was observed that the samples of ploughed soil also contained unevenly dispersed clumps of manure. The isolates characterised in the present investigation are listed in Table 1 according to the farm and source. The strains were identi ed as Typhimurium according to the Kau man^white typing scheme [13]. Phage typing was performed according to the scheme described by Callow [14] and modi ed by Anderson et al. [15] Pulsed eld gel electrophoresis (PFGE) All isolates were typed using PFGE to investigate the relationship among di erent isolates. Preparation of total DNA and experimental set up were as described in [16] except that plugs had a nal agarose concentration of 0.7%, and 0.1 mg ml 31 proteinase K was used for proteolysis. Restriction enzyme digestions were for 4 h, using either 20 U BlnI or XbaI enzyme in 50 Wl restriction bu er (Amersham Life Science, Buckinghamshire, UK) after which plugs were pre-incubated in the appropriate bu er at 37³C for 30 min. Electrophoresis was performed in a CHEF-DR III electrophoresis system (Bio-Rad Laboratories) at 14³C using 0.5UTBE running bu er and 1.1% Seakem 0 GTG 0 agarose gel (FMC Bioproducts, Rockland, ME, USA). Electrophoresis conditions were, for BlnI: phase one, initial switch time 8.0 s, nal switch time 20.0 s for 16 h, phase two, initial switch time 30.0 s, nal switch time 40.0 s for 3 h. For XbaI digestion the conditions were: phase one; initial switch time 17.0 s, nal switch time 26.0 s for 17.0 h; phase two, initial switch time 50.0 s, nal switch time 60.0 s for 4.5 h. Both PFGE programs were performed using 7.0 V cm 31 and a 120³ angle. The molecular size marker used was the Lambda Ladder PFG Marker (New England Biolabs). For visualisation, the DNA the gel was stained in ethidium bromide (2 Wg ml 31 ) for 10 min, de-stained in water for 20 min and photographed with Polaroid lm on an UV transilluminator. 3. Results S. typhimurium was isolated from the farm 1 environment in samples collected from the piggeries, conveyor belt, the outer surface and inner layers of manure and soil treated with manure (Table 1). Typhimurium could be isolated from ploughed soil up to 2 weeks after spreading of manure while no salmonellae were isolated from untreated soil. Furthermore, Typhimurium were isolated from pig faecal, piggery and slurry samples of farm 3, but not from the untreated soil or soil spread with slurry. All the Typhimurium isolates from farm 1 were of phage type DTU288. Likewise, the single isolate from farm 5 also belonged to phage type DTU288. On the other hand, all isolates from farm 3 were of phage type DT12.

3 D. Sandvang et al. / FEMS Microbiology Letters 187 (2000) 21^25 23 Table 1 Typhimurium isolated from pigs and environmental samples from three Danish farms Farm No. Isolate No. Isolate designation Date Type of sample Phage type Mar 98 Manure, outer surface U Mar 98 Manure, inner surface U Mar 98 Piggery sample U Mar 98 Manure, outer surface U Apr 98 Treated soil U Apr 98 Treated soil U Apr 98 Treated soil U Apr 98 Manure, outer surface U Apr 98 Conveyor belt U Apr 98 Conveyor belt U Apr 98 Treated soil U Apr 98 Treated soil U Apr 98 Manure, outer surface U Apr 98 Manure, outer surface U Apr 98 Conveyor belt U Apr 98 Conveyor belt U Apr 98 Piggery sample U May 98 Manure, outer surface U May 98 Manure, inner surface U May 98 Conveyor belt U July 98 Manure, inner surface U July 98 Conveyor belt U Nov 96 Clinical sample U Oct 96 Clinical sample U Aug 97 Clinical sample U June 98 Slurry July 98 Slurry U July 98 Slurry U July 98 Piggery sample U July 98 Piggery sample U Feb 97 Clinical sample U Oct 96 Clinical sample U Oct 97 Clinical sample U Dec 96 Clinical sample U288 PFGE analysis showed that all the isolates from farm 1 had identical PFGE patterns with restriction enzymes BlnI and XbaI (Fig. 1). PFGE patterns of the clinical isolate from farm 5 were identical to those of farm 1. Typhimurium isolated from the farmhouse environment and pigs in farm 3 were indistinguishable by PFGE using the above restriction enzymes. However, the PFGE patterns of farm 3 isolates di ered in more than 11 bands when compared with isolates from farms 1 and Discussion Fig. 1. PFGE typing of Typhimurium strains from farms 1 and 5. Lanes 1 and 14 contain molecular size markers (kb) (Lambda Ladder PFG Marker, New England BioLabs). Lanes 2^9, DNA from Typhimurium isolates from environmental samples of farm 1; lanes 10^12, the three sub-clinical samples from farm 1; lane 13, the porcine sub-clinical sample from farm 5. All Typhimurium DNA samples were digested with XbaI. Typhimurium was isolated from porcine faecal samples from farm 1 in October and November 1996 and again in August The same clone was identi ed in the environment during the spring and summer of 1998 although there was no clinical disease in pigs at that time. In samples taken from the piggeries, conveyor belt, manure and soil spread with manure, the same Typhimurium clone was identi ed approximately 20 months after rst isolation. This clone might both persist and survive in the farmhouse

4 24 D. Sandvang et al. / FEMS Microbiology Letters 187 (2000) 21^25 environment or in pigs with sub-clinical infection which excrete the bacteria. The similarity of PFGE patterns among farm 1 and farm 5 isolates indicates that the clone was introduced to farm 1 from the piglet producer farm 5. On the other hand, transfer of infection from farm 1 to farm 5 cannot be excluded since the same phage type of Typhimurium was isolated from clinical samples of farm 1 in October and November Farms 1 and 5 are close to each other, and other routes of transmission of Salmonella (including social contact between the two farmers, pets, birds, rodents, etc.) cannot be ruled out. According to Tenover et al. [17] bacterial isolates that are indistinguishable by PFGE are unlikely to demonstrate substantial differences by other typing techniques. This corresponds well with reports by other groups who also used PFGE to identify epidemiological and clonal relationships within Salmonella [6,18]. In this study, isolates from sub-clinically diseased animals and those taken from the environmental samples had identical phage types and were indistinguishable by PFGE. This correlates well with the ndings of Mutalib et al. [9] that the same Enteritidis phage types were found in samples from clinically diseased animals and in the poultry environment. These authors also identi ed isolates with the same phage type in rodents and birds, as those found in the environment. This emphasises the importance of rodents as possible transmission pathways or as sites of bacterial persistence, even when the farm animals have been eliminated and disinfection carried out. The re-isolation of the Typhimurium strain from farm 1 and farm 3 indicates that the strain survives in the farm environment resulting in a recrudescence of infection. Disposal of animal faeces with straw and organic waste makes the manure a solid mass, which may provide microenvironments for pathogens to survive, and not be distributed uniformly on the farmland. Thus, at farm 1 Typhimurium could be found in soil samples 14 days after spreading the manure, but at farm 3, which uses a slurry disposal system that spreads faecal material more uniformly on the elds, we were unable to detect Typhimurium from the soil spread with slurry. The di erence in management of pig faecal waste might play an important role in the survival of Salmonella especially since farm 1 repeatedly encounters Typhimurium in the farm environment at sub-clinical levels. However, more detailed studies are needed to investigate the signi cance of waste disposal systems. The die-o of pathogens like Salmonella in slurry and disinfection methods have been discussed previously by Heinonen-Tanski et al. [19] who described the advantages of aeration of slurry in pathogen reduction, even at low temperatures. Likewise, the comparison of Salmonella survival in composted and uncomposted animal manure has shown a signi cant reduction of the survival of salmonellae in composted manure [20]. Neither of the two farms investigated in this study employs any speci c disinfection measures in their handling of manure. Our investigations corroborate previous studies showing that a single clone of Typhimurium could persist on farm premises for a long time [11,12]. Thus, the persistence of Typhimurium strains in piggeries and on the elds should give rise to further control measures related to the handling of manure and disinfection of pig production facilities. Acknowledgements This project is nanced by a grant from the Danish Ministry of Food, Agriculture and Fisheries (MIL-97). Co-operation extended by the respective farmers is gratefully acknowledged. We would also like to thank Silvija Trajcevska and Lise Christensen for technical assistance and Dr. Kristian MÖller for his critical comments. References [1] Threlfall, J.E., Rowe, B. and Ward, L.R. (1993) A comparison of multiple drug resistance in salmonellas from humans and food animals in England and Wales, 1981 and Epidemiol. Infect. 111, 189^197. [2] Anonymous (1998) Annual Report on Zoonoses in Denmark [3] Davies, P.R., Morrow, W.E., Jones, F.T., Deen, J., Fedorka-Cray, P.J. and Harris, I.T. (1997) Prevalence of Salmonella in nishing swine raised in di erent production systems in North Carolina, USA. Epidemiol. Infect. 119, 237^244. [4] Baggesen, D.L., Wegener, H.C., Bager, F., Stege, H. and Christensen, J. (1996) Herd prevalence of Salmonella enterica infections in Danish slaughter pigs determined by microbiological testing. Prev. Vet. Med. 26, 201^213. [5] Christensen, J., Baggesen, D.L., Nielsen, A.C. and Nielsen, B. 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