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Title: Molecular detection of the index case of a subclinical salmonella Kentucky epidemic on a dairy farm

item Haley, Bradd
item ALLARD, MARC - Us Food & Drug Administration (FDA)
item BROWN, ERIC - Us Food & Drug Administration (FDA)
item HOVINGH, ERNEST - Pennsylvania State University
item Karns, Jeffrey
item Van Kessel, Jo Ann

Submitted to: Epidemiology and Infection
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2014
Publication Date: 5/22/2014
Citation: Haley, B.J., Allard, M., Brown, E., Hovingh, E., Karns, J.S., Van Kessel, J.S. 2014. Molecular detection of the index case of a subclinical salmonella Kentucky epidemic on a dairy farm. Epidemiology and Infection. DOI: 10.1017/S.0950268814001289.

Interpretive Summary: Salmonella enterica subsp. enterica is a fairly common pathogen known to infect man and animals. There are over 2500 serotypes of this organism and they vary greatly in their abilities in to colonize various hosts and in the degree of severity of disease they can cause. A dairy farm in Pennsylvania was monitored over an 8 year period for the presence of Salmonella in the cows and environment. During this period serotype Kentucky was detected in 2005 and again in early 2006. From 2006 until the end of the study in 2012 serotype Kentucky was epidemic in the lactating cows on the farm. Infection by S. Kentucky did not cause any detectable disease symptoms in the cows. DNA sequencing of the strain of Kentucky isolated from the farm in 2004 and a strain isolated in 2010 revealed several differences between the two. PCR assays were developed to detect those differences and they were used to examine many isolates of S. Kentucky obtained from the farm over the 7 year study period. The results showed that the strain of Kentucky found on the farm in 2004 was unable to persist in the cows or in the farm environment. The strain detected on the farm in 2006 was apparently introduced through a heifer returning from an off-site heifer raising facility in late 2005 and that, once introduced, it was epidemic in the lactating herd for the remaining 6 years of the study. Comparisons of the genomes of human pathogens that can persist in food animal production systems can lead to identification of factors that facilitate their persistence and to the development of methods that reduce their presence on the farm. Such reduction of pathogens in farm animals will lead to reduction in the risk of human infection through contaminated food products. This information will be useful to other scientists and regulatory agencies.

Technical Abstract: Salmonella enterica subsp. enterica serovar Kentucky, frequently isolated from poultry in the United States, is also commonly isolated from the feces of dairy cows and is an infrequent pathogen of humans. Using enrichments in tetrathionate broth followed by isolation on XLT4 agar, this serotype was sporadically isolated from environmental samples and fecal grab samples from dairy cows during routine sampling of a dairy farm in central Pennsylvania from 2004 to 2006. From 2006 to 2010 an asymptomatic S. Kentucky epidemic occurred during which the majority of cows (n ˜ 100 ± 10) were infected. To further investigate the epidemiology and molecular mechanisms of the onset and stasis of the epidemic, two S. Kentucky genomes were sequenced using 454 Roche technology; one from a farm isolate that was collected prior to the epidemic (2004) and one from an epidemic isolate (2010). Comparative genomic analysis demonstrated significant polymorphisms between the two strains. PCR primers targeting unique and strain-specific insertions/deletions were developed and screening of the archived isolates identified the index case of the asymptomatic epidemic as a heifer that was raised off-site and transported onto the study farm in 2006. Deeper investigation of the two isolates revealed 10 indels in open reading frames (ORFs) and 102 non-synonymous substitutions in protein-coding genes in the genomic DNA and putative plasmid DNA. Of these 102 amino acid polymorphisms, two have been identified as involved in S. enterica colonization of the bovine intestine. Further, an extra virulence repressor (haemolysin expression modulating protein) was identified on a ca. 70 kb IncI1 plasmid. We hypothesize that the virulence factors, namely Salmonella pathogenicity islands 1 and 2 (SPI1 and 2) are repressed in a manner that does not induce an immune response in the host, thereby allowing cells of this strain to colonize the cattle for long periods in a commensal relationship. This analysis demonstrates that intraserovar diversity accounts for the epidemiology and mechanisms of Salmonella dynamics and shows the utility of next generation genome sequencing in outbreak source detection.