FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK

1. FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK Cornell University Zoonoses Research Unit PI: Yrjö T. Gröhn, DVM, PhD Program Officer: Robert Hall, PhD April 3, 2008 http://www.people.cornell.edu/pages/ytg1/ http://www.vet.cornell.edu/popmed/

2. FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK Currently Cornell has two completed and four projects underway 1. Salmonella: 1.1. Transmission of MDR Salmonella - ZC001-03 PD – Professor Lorin Warnick Co - PD – Professor Martin Wiedmann 1.2.Sources and transmission of MDR Salmonella strains that cause human infection-ZC006-07 PD – Professor Lorin Warnick Co - PD – Professor Martin Wiedmann 2. Molecular Diagnosis: 2.1.Molecular Diagnosis of Bacterial Pathogens - ZC002-03 PD – Professor Yung-Fu Chang 2.2. C. difficile: Comparative Genomics and Strain Differentiation- ZC005-06 PD – Professor Yung-Fu Chang 3. Evolutionary Genomics: 3.1.Molecular Evolution of Campylobacter Diversity - ZC003-05 PD – Professor Michael Stanhope 4. Host Microbiota: 4.1.The Role of the Host Microbiota in Enteric Disease Development - ZC004-06 PD - Professor Craig Altier

3. FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK 1.1.Transmission of MDR SalmonellaProject No. ZC001-03 PD: Lorin Warnick http://www.popmed.vet.cornell.edu/bios/warnick.asp Co PD: Martin Wiedmann http://www.foodscience.cornell.edu/faculty/wiedmann.htm Co-investigators: Yrjo Grohn, Pat McDonough, Ynte Schukken Project Aims: • Determine key factors which determine the course of clinical outbreaks of (MDR) Salmonella in dairy cattle (industry funding). • Develop infectious disease transmission models and estimate transmission parameters for MDR Salmonella • Evaluate the relationships among MDR Salmonella isolates from cattle and human samples

4. Why Salmonella? • Leading human foodborne pathogen (more deaths than any other known human foodborne pathogen) • Estimated 550 deaths among 1.4 million cases annually in the US • No clear decline in human salmonellosis infections despite decline in infections with other foodborne pathogens • Emergence and spread of multi-drug resistant Salmonella is a concern • More severe illness and increased risk of hospitalization • Dairy cattle may be important source of MDR Salmonella (e.g. Newport, Typhimurium, 4,5,12:i:- , and Dublin)

5. ST 25 ECO157H7 ST 75 90 ST 77 ST 1 ST 2 ST 3, 14, 22*, 23, 26, 50, 82, 83 ST 15* ST 24* ST 36 ST 79 ST 48 75 ST 18 ST 81 ST 19, 20*, 21 ST 5* 6, 47, 49, 51 64 ST 7 Newport Type B (humans, birds) ST 8 ST 42, 43 53 ST 27 ST 12* ST 13, 46, 70, 76, 78 ST 17, 34 54 ST 73 ST 41 ST 39, 84 ST 16* ST 29 ST 28 ST 30 ST 4, 9 ST 37 ST 44 63 ST 32 ST 35, 74 Newport Type A (cattle and humans, often MDR; ST31 is an S. Litchfield isolate) ST 11, 31, 85 82.8 ST 33 82 ST 38 ST 80 ST 10* 53 ST 40 ST 45 Aim 1.1.1: Field Project Highlights • 10% of herds per year had laboratory-confirmed salmonellosis • Typhimurium and Newport accounted for >50% of affected herds • Both types frequently associated with multi-drug resistance • Identified emerging serotype 4,5,12:i:- in several herds • Results suggest that herds with clinical outbreaks represent most risk for human exposure to MDR Salmonella • Salmonella rarely isolated from non-clinical control farms • Shedding persists long after recovery • Subtyping Salmonella isolates from cattle and humans identified both overlapping and distinct populations • MDR Newport, Typhimurium, and 4,5,12:i:- were common to both

6. Duration of Fecal Salmonella Shedding Following Clinical Disease in Animal Hosts Implications of continued shedding: • Environmental contamination • Within-herd transmission • Transmission to other herds • All of the above lead to increased risk of transmission to humans

7. Results: Serotype

8. Results: Age Group

9. Results: Summary • Fecal Salmonella shedding often persists beyond clinical outbreak in herd and may exceed 1 year • The proportion of animals shedding for at least 2 months was significantly higher in adult cows than calves (Fisher’s exact test p-value=0.008) • Results from these 22 herds improve on data from past studies in relatively few outbreak herds • Provides parameter estimates for Salmonella transmission modeling

10. INFECTION CONTROL Control at the human population (e.g. hygiene, human vaccination) HUMAN HEALTH INFECTION RESERVOIR Aim 2: Highlights in Mathematical Modeling Control at the transmission routes (e.g. heat treatments) Control at the infection reservoir

11. Using mathematical modeling approach … To understand the ecology, emergence and spreadof MDR Salmonella in infection reservoirs and to help designing effective control strategies. Specifically, we address… • How the heterogeneity in host infectiousness affects the dynamics of transmission and the efficacy of control strategies within the infection reservoir. • Dynamics of infection in small transient populations.

12. 1. Heterogeneity in host infectiousness • Clinically infected individuals were the main force of infection transmission. • Subclinically infected individuals with long infectious period but low contagiousness had a small impact on transmission. • High efficacy vaccines were necessary to eradicate infection. • The presence of super-shedders made necessary the application of strategies targeting this specific group rather than population-wide control strategies. *Lanzas C. et al, The effect of heterogeneous infectious period and contagiousness on thedynamics of Salmonella transmission in dairy cattle, Epidemiology and Infection,2008, 136, 1496-1510.

13. 2. Dynamics of infection in small transient populations • Enteric multidrug resistant pathogens transmit effectively in small populations (e.g. health care facilities, farms). • Large fluctuations in the prevalence of infection happen by chance and infections have a large probability of extinction. • Extensive research in infection control practices, but little research on the underlying dynamics of infection… What favors transmission?

14. Dynamics of infection in small transient populations: modeling approach • Development of an indirect transmission model for enteric multidrug resistant pathogens. • Study of the infection dynamics without infection control (salmonellosis in a calf-rearing operation as a case study*). • Evaluation of intervention strategies to control infection. • Development of a theoretical framework *Lanzas C., et al., The risk and control of Salmonella outbreaks in calf-rearing operations, Veterinary Research, 2008: 39:61.

15. Mathematical model of indirect transmission

16. Differential equations

17. Factors that favor infection persistence in small populations • High turnover rates of the system (continual replenishment of the susceptible pool) • Continual admission of infected individuals from the ‘community’ level • Environmental reservoirs

18. (a) 1000 800 600 number of cases 400 200 0 base adm50 adm100 imm75 imm37 imm20 shed30 shed60 cont33 cont66 hyg33 hyg66 scenario 1500 (b) 1000 duration of outbreak (days) 500 0 base adm50 adm100 imm75 imm37 imm20 shed30 shed60 cont33 cont66 hyg33 hyg66 scenario Intervention strategies to control infection Most effective strategies: Assigning workers/ equipment to groups of individuals Complete close of the facilities to incoming individuals Immunization of a high proportion of admitted individuals Hygiene Without interventions

19. FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK 1.2. Sources and transmission of MDR Salmonella strains that cause human infection Project No. ZC006-07 PD: Lorin Warnick http://www.popmed.vet.cornell.edu/bios/warnick.asp Co-investigators: Yrjo Grohn, Pat McDonough, Martin Wiedmann Joint project with WSU ZRU

20. FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK Project Aims: • Subtyping methods for highly clonal MDR Salmonella • Retrospectively characterize the distribution and transmission of MDR Salmonella in the northeastern and northwestern U.S. • Prospectively monitor evolution and emergence of MDR strains • Identify risk factors for human acquisition of MDR Salmonella • Define targets for control strategies through field studies

21. Salmonella serotype 4,5,12:i:- is a major emerging Salmonella serotype • A monophasic variant of Salmonella Typhimurium • First been reported in the literature in 1993 (Thailand) • Prevalence of serotype 4,5,12:i:- among human salmonellosis cases has increased considerably over the last 10 – 15 years • In the U.S., serotype 4,5,12:i:- represented 0.2 % and 2.3% of human clinical isolates in 1995 and 2005, respectively. • Responsible for human salmonellosis outbreak linked to poultry pot pies in 2007.

22. Aim 1: Characterization of Salmonella serotype 4,5,12:i:- • Developed a collection of 102 Salmonella Typhimurium and 92 Salmonella 4,5,12:i:- isolates from different sources (human, animal, and food) and geographical locations (US [NY, GA, WA] and Spain) • Characterized isolates by different subtyping methods (MLST, PFGE, PCR screens for presence/absence of selected genes, including genes responsible for phase 2 flagella expression)

23. Aim 1: Highlights - Evolution and emergence of Salmonella serotype 4,5,12:i:- • Salmonella 4,5,12:i:- isolates from Spain and the US appear to largely represent distinct clones with distinct gene deletion patterns • One US isolate matches the “Spanish clone” • Salmonella 4,5,12:i:- represents multiple independent emergence events, including the common US and Spanish clones as well as additional rare 4,5,12:i:- genotypes • Future efforts will focus on (i) understanding worldwide distribution of different 4,5,12,i:- clones, (ii) the evolution of antibiotic resistance in 4,5,12i:- strains, and (iii) probing the potential selective advantage of a loss of phase 2 flagella expression.

24. Aim 2: Retrospective molecular subtyping of human and animal Salmonella isolates • Characterized 157 clinical Salmonella isolates from cattle and 178 clinical Salmonella isolates from humans by serotyping and pulsed-field gel electrophoresis (PFGE) • 167 PFGE patterns, 116 patterns unique to human isolates, 44 unique to cattle isolates; 7 patterns found among both human and cattle isolates • Subtype data available in PathogenTracker • Among cattle isolates, three PFGE types were identified in multiple farms in adjacent counties, indicating geographical clustering of Salmonella subtypes

25. 1640 Salmonella isolates

28. Aim 4: Case-case study • Objective: Identify risk factors for MDR Salmonella infections (Newport, Typhimurium, and 4,5,12:i:-) in people • In collaboration with NY State Department of Health • specifically, we will determine the relative importance of foodborne exposure and direct contact for MDR Salmonella transmission • Patients infected with Salmonella isolates matching bovine strains by serotype, antibiotic resistance, and PFGE profile will be compared to patients with Salmonella isolates that are pan-susceptible and not associated with cattle

29. Case-case Study… • Data collected by the NYSDOH from Emerging Infections Program counties • Currently have questionnaire data and isolates representing 72 cases • Enteritidis: 17% (12) • Typhimurium: 17% (12) • Newport: 9% (6) • Tennessee: 7% (5) • Heidelberg: 4% (3) • Thompson: 4% (3) • 20 other serotypes

30. Aim 5: Field Study • Objective: Determine the effect of clinical Salmonella outbreaks in dairy cattle on the prevalence of MDR Salmonella fecal shedding and environmental contamination • This will help guide control strategies for reducing the public health threat of bovine salmonellosis • For example, can high risk herds be recognized by clinical laboratory accessions or is surveillance required in herds without clinical disease? (See poster for preliminary results)

31. FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK 2.1. Molecular Diagnosis of Bacterial Pathogens Project No. ZC002-03 PD – Professor Yung-Fu Chang http://www.popmed.vet.cornell.edu/bios/chang.asp The overall goal of this project is to: Develop a multi-pathogen identification microarray for high confidence identification of food- and water-borne pathogens based on their virulence factors as probes. Work completed

32. Publication • Ku et al. 2005. Identification and characterization of in vivo attenuated mutants of Salmonella enterica serovar Choleraesuis using signature-tagged mutagenesis in a pig infection model. Infect. Immun. 73:8194-8203. • Palaniappan et al. 2006. Differentiation of Escherichia coli pathotypes by oligonucleotide spotted array. J. Clin. Microbiol. 44:1495-1501. • Yu et al. 2007. Prevalence and characterization of multidrug-resistant (ACSSuT) Salmonella enterica serovar Typhimurium isolated from our Gosling’s farms and a hatchery farm. J. Clin. Microbiol. 46:522-526. • Scaria et al. 2008. Microarray for molecular typing of Salmonella enterica serovars. Mol. Cell Probes 22:238-243. • Scaria et al.Microbial Diagnostic Array Workstation (MDAW): A Web Server for Diagnostic Array Data Storage, Sharing and Analysis. Source Code Biol. Med. 3:14-18. • Scaria et al. Development of a microarray for detection of antimicrobial genes. In preparation.

33. FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK Clostridium difficile:genomic and transcritome studies Project No. ZC005-06 PD – Professor Yung-Fu Chang http://www.popmed.vet.cornell.edu/bios/chang.asp

34. Clostridium difficile A spore-forming, gram-positive bacillus that produces exotoxins that are pathogenic to humans C. difficile-associated disease (CDAD) ranges in severity from mild diarrhea to fulminant colitis and death Antimicrobial use is the primary risk factor for development of CDAD because it disrupts normal bowel flora and promotes C. difficile overgrowth

35. Objectives 1. To perform a comparative genomic study of C. difficile and establish a microarray database 2. To develop a specific diagnostic microarray for diagnosis of Clostridial species 3. To study the transcriptome/proteome of C. difficile grown in vitro and in vivo

36. Comparative genomics of Clostridium difficileOverview of the microarray results Equine Bovine Swine Food Human

37. Core genes

38. Flagella-related genes Eq Bv Sw Fd Human

39. Virulent genes Eq Bv Sw Fd Human

40. Antibiotic resistance genes Eq Bv Sw Fd Human

41. PFGE pattern Eq Bv SwFd Human

42. On-going works 2. Development of a diagnostic array for Clostridial species 3. Study of the transcriptome/proteome of C. difficile during its infection in vivo using a pig model as a host

43. FOOD & WATERBORNE DISEASES INTEGRATED RESEARCH NETWORK 3.1. Evolution of Campylobacter diversityProject No. ZC003-05PD – Professor Michael Stanhopemjs297@cornell.edu • Project start date: 04/01/06 • Campylobacter - most common bacterial cause of foodborne illness in USA and much of the developed world Two species of particular importance: C. jejuni and C. coli • Sheep, cattle, pigs, and poultry - both C. jejuni and C. coli; C. jejuni predominates in bovine and chicken - C. coli in swine and turkey

44. Evolution of Campylobacter diversityIntroduction… • Principal overall goal of this project: • provide detailed understanding of genetic diversity and molecular adaptation of Campylobacter coli and Campylobacter jejuni in relation to their different hosts • corollary purpose: assess whether there are particular animal reservoirs and/or strains serving as more likely sources of human infection and antibiotic resistance.

45. Expanded MLST scheme: 16 genes; 70 C. coli isolates, from swine, chicken, bovine, and human.Sequence types (ST) and clonal complexes identified based on Campylobacter MLST database (http://pubmlst.org/campylobacter/)

46. Analysis of 16 gene MLST Genotypic similarity defining clonal complex = 13/16 shared alleles Genotypic similarity defining clonal complex = 12/16 shared alleles Fisher exact test; null hypothesis of independence of genotype and host: P<0.001 Fisher exact test; null hypothesis of independence of genotype and host: P<0.001 Bovine Human Chicken Swine => Evidence supporting C. coli host specific ecotypes

47. Genome Adaptation • gene presence/absence • molecular adaptation of protein coding genes • gene regulation

48. Microarrays • Genome sequence allows gene presence / absence detection across strains using microarrays • E.g. Combimatrix 4 X 2K microarrays

49. Swine Bovine Chicken Dendrogram and heatmap of variable genes among 36 C. coli test strains using oligonucleotide microarray• sets of genes common to isolates derived from different hosts - human isolates currently under experimentation

50. Ancestral genome reconstruction • Ancestral genomes reconstructed from composition of interspecies genome comparisons; • provides assessment of gene/presence absence in an evolutionary context • Gene gain, loss and duplication on each lineage