Salmonella Typhimurium

Introduction

Salmonellosis is a globally distributed foodborne disease significant impact on public health. More than 2500 serotypes of Salmonella have been identified (4) and Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Heidelberg are the most prevalent serotypes (9).

Salmonella Typhimurium is the second most common serotype responsible for causing human salmonellosis in Denmark (1). Poultry and poultry related products are considered as important sources of human salmonellosis with Salmonella Typhimurium (2). The prevalence of Salmonella in poultry is very low in Denmark over quite a long period. However, multiple outbreaks of Salmonella Typhimurium DT 41 have occurred in some broiler breeder farms. Out of 19 broiler breeder flocks, 13 were found positive for this specific phage type (6).

Information concerning the epidemiology of DT 41 is scanty, but it has been isolated from wild birds (5, 7, 8) indicating that wild birds could be a source of this phage type. Presence of DT 41 in Danish broiler flocks has been described and that major genetic diversity occurs among the isolates (6).

Persistence or reintroduction of this phage type into the broiler breeder flocks could be responsible for the problems observed. Additionally, environmental factors like poultry related vehicles or host factors might add to the DT 41 problems. The objective of this study was to investigate the genomic stability of Salmonella Typhimurium DT41 following in-vitro and in-vivo experiments.

Material and methods

In-vitro study
Four strains of DT41 were used in this study to investigate the genomic stability under different temperatures. The pelleted poultry feed and dust particles were considered as two vehicles. Tubes containing pellet and dust were inoculated with a concentration of 108 CFU/ml, sealed thereafter and stored at three different temperatures: 5˚C, 20˚C and 41˚C. Finally inoculated tubes were examined periodically to test the viability of DT41 following standard bacteriological procedures. In-vivo study Birds of three different age groups - 30, 40 and 60 weeks-old were placed separately in three different boxes. Each age group consisted of 11 birds. Two isolates of DT41 were used in this study - one was poultry origin which also was used in in-vitro study while the other one was a human isolate. The challenge inoculum was prepared from the overnight broth culture and all the birds were inoculated orally with a concentration of 108 CFU of bacteria per ml. To assess faecal excretion of DT41, cloacal swabs  were collected everyday for the first week from each bird and then weekly intervals. The birds were observed for 3 weeks from the day of primary infection. At the termination of the experiment, the birds were sacrificed and subjected to post-mortem examination to observe any gross pathological changes. Caecal tonsils from each bird were collected. Both cloacal swabs and caecal tonsils were checked for Salmonella following classical bacteriological procedures. Genetic stability of cultures obtained was investigated by PFGE (3) and MLVA (10).

Results

In-vitro experiment
The pellet tubes incubated at 5°C revealed that 3 strains of DT41 survived for 12 weeks and only one strain survived for 18 weeks. DT41 was recovered from the tubes kept at 20°C until 30 weeks. At temperature 41°C, all of the four strains survived only for 2 weeks.

In case of dust particles, none of the four strains survived even for a week at 41°C. But at 5°C, 2 strains were recovered at week 2 and 3 each and only one strain was recovered until 3 weeks at 20°C. Preliminary results of PFGE and MLVA showed that some genetic changes have occurred in some of the strains.

In-vivo experiment
The number of birds excreting DT41 in each group varied considerably between sampling dates. The highest percentage of birds excreting DT41 was found in birds 60 weeks of age and the excretion rate gradually decreased after 7 days in each of the three groups. Although not statistically significant, birds of older age groups, i.e. 60 weeks of age were proportionately higher than other two groups in terms of rate of recovery.

In case of the human isolate, after 21 days of repeated cloacal samples examination, it was found that the excretion rate was significantly different between 30 and 60 weeks of age groups of birds at day 14. No genetic changes have been observed in the isolates based on preliminary results of PFGE and MLVA.

Discussion

This study provides an insight into the dynamics of the stability of the genome of DT 41 under different temperature conditions and in different vehicles as well as generates information on colonization and subsequent excretion of Salmonella Typhimurium DT41 from chickens infected with DT 41 isolates of poultry and human origins. In-vitro experiment revealed that the strains did not survive in dust particles more than 3 weeks, considering all the temperature categories, but in case of pellets at temperature 20°C, DT41 survived more than 6 months. 

In case of dust samples, when results were negative, multiple tubes were examined to increase the chances of recovering the bacteria and to confirm the absence of growth. In addition, dust samples were also tested for the presence of any antibacterial substances.

In in-vivo experiment it has been observed that the rate of persistency of DT41 might be much higher in aged birds if infected with DT41. This finding reconfirms the findings of Litrup et al (6) that, birds of older age groups are more prone to become infected with DT41. However, this remains unexplainable from this study, but nonspecific host factors might be responsible for this age variation.

Many factors might play roles in this result observed in the experimental study. Answers to some questions might be revealed after analyzing the isolates genotypically after exposure to different environmental as well as host factors.

References

1. Anon, 2005. Annual report on zoonoses in Denmark 2004. Copenhagen: Ministry of Food, Agriculture and Fisheries. 

2. Baggesen, D. L., and Wegener, H. C. 1994. Phage types of Salmonella enterica ssp. enterica serovar XVII WVPA Congress. Cancun, Mexico 216 Typhimurium isolated from production animals and humans in Denmark. Acta Vet. Scand. 35:349-354.

3. Centers for Disease Control and Prevention (CDC). 2009. One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Escherichia coli O157:H7, Salmonella serotypes, and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE). 

4. Grimont, P. A. D., and Weill, F-X. 2007. Antigenic Formulae of the Salmonella Serovars (9th edition). WHO Collaborating Centre for Reference and Research on Salmonella. Institut Pasteur, Paris, France. Available at: http://www.pasteur.fr/ip/portal/action/WebdriveActionEvent/oid/01s-000036-089

5. Hughes, L. A., Shopland, S., Wigley, P., Bradon, H, Leatherbarrow, A. H., Williams, N. J., Bennett, M., de Pinna, E., Lawson, B., Cunningham, A. A. and Chantrey, J. 2008. Characterization of Salmonella enterica serotype Typhimurium isolates from wild birds in Northern England from 2005 - 2006. BMC Vet Res. 4:4.

6. Litrup, E., Christensen, H., Nordentoft, S., Nielsen, E. M., Davies, R. H., Helmuth, R., and Bisgaard, M. 2010. Use of multiple-locus variable-number tandem-repeats analysis (MLVA) typing to characterize Salmonella Typhimurium DT 41 broiler breeder infections. J Appl Microbiol. 109 (6):2032-2038.

7. Pennycott, T. W., Cinderey, R. N., Park, A, Mather, H. A. and Foster, G. 2002. Salmonella enterica subspecies enterica serotype Typhimurium and Escherichia coli 086 in wild birds at two garden sites in south-west Scotland. Vet Rec. 151:563-567.

8. Pennycott, T. W., Park, A., Mather, H. A. 2006. Isolation of different serovars of Salmonella enterica from wild birds in Great Britain between 1995 and 2003. Vet Rec. 158: 817-820.

9. Schlundt, J., Toyofuku, H., Jansen, J. and Herbst, S. A. 2004. Emerging foodborne zoonoses. Rev Sci Tech. 23(2). 513-533. Torpdahl, M., Sørensen, G., Lindstedt, B. A. and Nielsen, E. M. 2007. Tandem repeat analysis for surveillance of human Salmonella Typhimurium infections. Emerg Infect Dis. 13:388-395.