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/
1
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
2
FOOD & WATERBORNE DISEASES INTEGRATED
RESEARCH NETWORK
1.1.Transmission of MDR Salmonella
Project 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:
1. Determine key factors which determine the course of clinical
outbreaks of (MDR) Salmonella in dairy cattle (industry funding).
2. Develop infectious disease transmission models and estimate
transmission parameters for MDR Salmonella
3. Evaluate the relationships among MDR Salmonella isolates from
cattle and human samples
3
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)
4
Aim 1.1.1: Field Project Highlights
•
•
•
•
•
10% of herds per year had laboratoryconfirmed 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
ST 25
ECO157H7
ST 75
ST 7790
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
ST 8
ST 42, 43
ST 27
53
ST 12*
ST 13, 46, 70, 76, 78
Results suggest that herds with clinical
outbreaks represent most risk for human
exposure to MDR Salmonella
– Salmonella rarely isolated from nonclinical control farms
– Shedding persists long after recovery
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
ST 32
63
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
Newport Type B
(humans, birds)
ST 35, 74
82.8
82
ST 38
ST 80
ST 10*
ST 40
53
ST 45
ST 11, 31, 85
ST 33
Newport Type A
(cattle and humans,
often MDR; ST31 is
an S. Litchfield
isolate)
5
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
6
Results: Serotype
Serotype
4,5,12:i:Infantis
Kentucky
Newport
Typhimurium
Other
% (N)
Mean duration
(days)
Max duration
(days)
9.0% (32)
31
183
10.4% (37)
18
84
4.8% (17)
44
245
51.0% (182)
32
391
8.4% (30)
18
66
16.5% (59)
35
388
7
Results: Age Group
Age Group
Female calves
Adult cows
% (N*)
Mean duration
(days)
Max duration
(days)
20.8% (73)
19
72
76.6% (269)
34
391
*Excluding 15 with missing or “other” age group
8
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
9
Aim 2: Highlights in Mathematical Modeling
INFECTION CONTROL
HUMAN
HEALTH
Control at the human population
(e.g. hygiene, human vaccination)
Control at the transmission routes
(e.g. heat treatments)
INFECTION
RESERVOIR
Control at the infection reservoir
Using mathematical modeling approach …
To understand the ecology, emergence and spread of
MDR Salmonella in infection reservoirs and to help
designing effective control strategies.
Specifically, we address…
1. How the heterogeneity in host infectiousness
affects the dynamics of transmission and the
efficacy of control strategies within the infection
reservoir.
2. Dynamics of infection in small transient
populations.
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 the dynamics of
Salmonella transmission in dairy cattle, Epidemiology and Infection, 2008, 136, 1496-1510.
2. Dynamics of infection in small transient
populations
30
25
Number of infective individuals
• 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?
20
15
10
5
0
0
10
20
30
40
Time
50
60
70
80
Dynamics of infection in small transient
populations: modeling approach
1. Development of an indirect transmission
model for enteric multidrug resistant pathogens.
2. Study of the infection dynamics without
infection control (salmonellosis in a calfrearing operation as a case study*).
3. Evaluation of intervention strategies to
control infection.
4. 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.
Mathematical model of indirect transmission
INDIVIDUALS
SUSCEPTIBLE
INFECTED
RECOVERED
WORKERS
UNCONTAMINATED
CONTAMINATED
Differential equations
dS
N
V
  (1    )(1  )  cpi
S   (t )qSW
dt
K
V
Infection
Exit

Admission
dI
N
V
 (1  )  cpi
S  (m   ) I   (t )q I W
dt
K
V
mortality
Recovery
dR
N
  (1 
)  I   (t ) q RW
dt
K
dV 
I 

 V  cpc V
dt
N
Decontamination Contamination
dV 
dV 

dt
dt
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
Intervention strategies to control infection
1000
Assigning workers/
equipment to groups of
individuals
number of cases
Most effective strategies:
Complete close of the
facilities to incoming
individuals
Hygiene
600
400
200
0
duration of outbreak (days)
Immunization of a high
proportion of admitted
individuals
800
base adm50 adm100imm75 imm37 imm20 shed30 shed60 cont33 cont66 hyg33 hyg66
scenario
1500
1000
500
0
base adm50 adm100imm75 imm37 imm20 shed30 shed60 cont33 cont66 hyg33 hyg66
scenario
Without interventions
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
19
FOOD & WATERBORNE DISEASES INTEGRATED
RESEARCH NETWORK
Project Aims:
1.
2.
3.
4.
5.
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
20
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.
21
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)
22
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.
23
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
24
1640 Salmonella isolates
25
26
27
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
28
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
29
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)
30
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
34
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.
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
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
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
1.
Comparative genomics of Clostridium difficile
Overview of the microarray results
Equine
Bovine Swine Food
Human
Core genes
Flagella-related genes
Eq
Bv
Sw Fd
Human
Virulent genes
Eq
Bv
Sw Fd
Human
Antibiotic resistance genes
Eq
Bv
Sw Fd
Human
PFGE pattern
Eq
Bv
Sw
Fd
Human
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
FOOD & WATERBORNE DISEASES INTEGRATED
RESEARCH NETWORK
3.1. Evolution of Campylobacter diversity
Project No. ZC003-05
PD – Professor Michael Stanhope
mjs297@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
46
Evolution of Campylobacter diversity
Introduction…
• 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.
47
Isolate
cco1
cco2
cco3
cco4
cco5
cco6
cco7
cco8
cco10
cco11
cco12
cco9
cco13
cco14
cco15
cco16
cco17
cco80
cco18
cco19
cco20
cco23
cco24
cco25
cco27
cco37
cco49
cco51
cco52
cco54
cco55
cco60
cco61
cco62
cco63
cco65
cco67
cco69
cco70
cco71
cco72
cco73
cco74
cco75
cco76
cco77
cco78
cco79
cco81
cco82
cco83
cco85
cco86
cco87
cco88
cco89
cco90
cco91
cco92
cco93
cco94
cco95
cco96
cco97
cco98
cco99
cco100
cco101
cco102
RM2228
C.jejuni
adk
1
1
1
1
1
1
1
1
2
1
2
1
2
2
1
2
1
8
3
2
2
1
2
2
4
5
2
2
2
2
1
5
1
5
5
5
1
1
2
6
7
6
6
7
7
1
1
2
5
1
2
6
7
9
1
10
1
5
1
1
1
1
11
1
1
1
6
1
1
2
12
aroE mdh nadP pheS
1
1
1
1
1
1
2
1
1
1
3
1
1
1
4
2
2
1
2
1
1
1
4
2
3
1
4
3
1
2
5
2
1
3
6
5
1
1
5
2
2
4
22
1
1
1
4
4
1
5
7
1
4
1
8
1
1
6
4
4
1
5
7
1
1
6
9
4
1
12
12
1
1
7
10
2
2
4
4
1
2
4
4
1
1
1
4
2
2
4
4
1
2
4
4
1
2
4
4
1
1
7
10
2
2
4
4
1
2
4
4
1
2
4
4
1
2
4
4
1
1
1
11
2
1
1
11
2
1
1
10
2
1
1
11
2
1
1
11
2
1
1
11
2
1
1
3
2
1
8
7
1
1
8
12
1
5
9
13
6
1
10
10
1
1
11
14
1
1
11
15
1
1
4
4
7
1
10
10
1
1
4
14
1
1
10
14
1
1
10
16
1
1
1
14
1
2
13
10
1
6
14
17
8
1
15
10
1
1
16
10
1
1
3
16
10
2
1
10
2
20
18
10
7
3
7
1
1
10
10
1
1
8
14
1
5
17
19
1
2
13
10
1
2
18
20
1
1
1
21
2
2
10
7
1
1
7
1
1
7
1
1
7
1
1
19
5
2
1
19
5
2
4
1
23
1
8
21
24
11
pgi
1
2
3
4
1
5
4
1
7
4
8
6
3
3
6
3
6
6
1
9
9
3
9
9
9
1
10
9
9
9
9
9
11
1
9
9
3
3
6
6
6
12
6
9
3
13
13
3
6
14
6
9
6
3
9
15
16
3
6
16
14
17
3
14
1
1
1
2
2
3
18
recA carB truM aspA
1
1
1
33
1
1
2
33
1
2
3
33
2
1
4
53
1
1
2
33
1
1
5
33
1
2
6
32
2
1
2
33
1
1
7
33
1
1
4
53
3
1
8
33
1
3
6
33
1
2
9
33
1
2
6
33
1
1
10
33
1
2
9
33
1
3
6
33
1
8
2
33
1
4
11
33
3
2
8
33
3
2
8
33
2
1
12
33
3
2
8
33
3
2
8
33
3
2
8
33
1
4
11
33
3
2
8
33
3
2
8
33
3
2
8
33
3
2
8
33
2
1
11
33
2
4
4
33
2
5
2
33
2
4
11
33
2
4
4
33
2
4
4
33
2
1
4
33
1
1
6
33
1
2
2
33
4
6
13
33
1
2
26
33
1
1
2
33
1
1
2
33
5
2
14
33
1
2
6
33
6
1
15
33
6
1
16
33
1
7
17
33
1
1
18
33
1
2
19
33
1
1
6
33
1
1
6
33
1
1
17
33
8
1
20
33
2
1
21
32
9
9
22
33
2
2
9
32
1
1
2
33
1
10
23
33
1
10
17
33
1
2
19
33
4
1
24
33
1
1
2
33
1
2
25
33
10
1
4
33
10
1
4
33
10
1
6
33
1
1
11
53
1
1
11
53
1
2
27
33
11
11
28
2
glnA
gltA
glyA
pgm
tkt
uncA
ST
Clonal complex
38
38
39
30
30
30
44
30
30
44
30
30
30
30
30
30
30
30
30
30
167
167
82
82
78
82
82
82
173
82
78
82
82
82
82
82
82
104
104
104
104
104
104
104
152
104
118
104
113
113
104
113
113
113
43
44
43
35
44
35
43
173
35
35
35
43
47
43
43
47
43
17
36
17
36
36
36
36
68
68
36
17
17
17
41
17
17
17
1467
NEW
828
NEW
NEW
890
1061
1102
NEW
1631
1113
829
825
1017
829
825
829
894
1436
1068
NEW
NEW
NEW
NEW
NEW
NEW
NEW
1068
NEW
NEW
1104
1104
NEW
NEW
NEW
NEW
1104
1585
2301
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
829
825
1191
1068
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
855
855
1586
ST-828 complex
ST-828 complex
ST-828 complex
889
2642
892
ST-828 complex
ST-828 complex
ST-828 complex
292
38
38
39
38
293
39
38
39
39
39
39
39
39
39
39
39
39
39
39
294
39
39
153
39
39
39
39
39
39
153
153
153
65
82
113
47
17
39
44
30
30
244
245
246
30
44
30
30
30
246
30
30
247
248
30
30
30
82
78
78
82
78
78
329
330
78
78
331
332
82
82
82
82
82
82
82
104
104
416
104
104
104
104
104
104
104
104
104
104
104
104
104
104
104
104
44
43
335
336
337
43
43
338
43
43
43
43
85
85
85
85
85
85
85
17
17
17
68
17
17
17
17
17
17
17
17
68
68
68
68
68
68
68
39
176
110
39
39
39
39
176
39
39
176
39
39
39
38
38
39
38
39
42
176
39
39
39
39
38
39
38
38
38
38
38
30
30
103
30
30
30
30
30
30
30
30
249
30
30
30
30
30
30
250
30
30
30
30
30
66
30
30
30
30
30
30
30
82
115
333
82
82
82
78
82
79
79
82
79
82
82
79
115
79
82
219
82
79
328
79
82
79
82
82
82
82
82
82
82
189
104
188
113
113
189
104
417
104
104
113
104
113
113
104
113
418
152
104
113
104
104
113
104
104
113
104
104
104
118
118
43
43
339
43
47
47
43
43
35
35
43
64
35
47
35
43
43
35
340
43
43
43
43
47
47
35
47
43
43
332
44
44
17
17
246
17
17
17
17
17
17
17
17
17
17
41
17
17
17
17
17
17
17
17
41
17
41
17
17
17
17
17
36
36
39
30
140
113
43
41
1
5
3
4
1
5
419
900
-
898
3020
3340
901
825
3348
1096
825
854
854
3336
1147
1147
1063
43
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-828 complex
ST-21 complex
Host
Swine
Swine
Swine
Swine
Swine
Swine
Swine
Swine
Swine
Swine
Swine
Chicken
Chicken
Chicken
Chicken
Chicken
Chicken
Turkey
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Bovine
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Chicken
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/campylob
acter/)
48
Analysis of 16 gene MLST
Genotypic similarity defining
clonal complex = 13/16 shared alleles
Genotypic similarity defining
clonal complex = 12/16 shared alleles
Clonal
Composition of clonal complex
complex
Clonal
complex
Composition of clonal complex
1
Cco19, Cco52, Cco54, Cco25,
Cco27, Cco24, Cco49, Cco20
1
Cco19, Cco52, Cco54, Cco25,
Cco27, Cco24, Cco49, Cco20
2
Cco60, Cco63, Cco65, Cco67,
Cco62
2
Cco60, Cco63, Cco65, Cco67,
Cco62
3
Cco 94, Cco82, Cco97
3
Cco 94, Cco82, Cco97
4
Cco9, Cco17, Cco15
4
Cco9, Cco17, Cco15
5
Cco37, Cco18
5
Cco37, Cco18
6
Cco5, Cco2
6
Cco5, Cco2
7
Cco78, Cco77
7
Cco78, Cco77
8
Cco74, Cco73
8
Cco74, Cco73
9
Cco76, Cco72
Fisher exact test; null hypothesis of
independence of genotype and host: P<0.001
Bovine
Human
Fisher exact test; null hypothesis of
independence of genotype and host: P<0.001
Chicken
Swine
=> Evidence supporting C. coli host specific ecotypes
Genome Adaptation
• gene presence/absence
• molecular adaptation of protein coding
genes
• gene regulation
50
Microarrays
• Genome sequence allows gene presence / absence detection
across strains using microarrays
– E.g. Combimatrix 4 X 2K microarrays
51
Bovine
Chicken
CCO14
CCO15
CCO9
CCO17
CCO6
CCO1
CCO7
CCO11
CCO4
CCO2
CCO5
CCO8
CCO62
CCO10
CCO49
CCO54
CCO24
CCO19
CCO20
CCO55
CCO61
CCO23
CCO67
CCO12
CCO27
CCO25
CCO52
CCO51
CCO3
CCO13
CCO16
CCO18
CCO37
CCO63
CCO60
CCO65
13
12
321
11
292
310
276
95
153
93
144
80
79
81
82
83
224
315
219
113
112
114
195
111
92
228
227
230
270
134
88
154
155
170
103
281
282
176
322
110
98
226
105
72
166
71
135
171
109
108
70
45
49
68
47
97
46
124
125
126
127
151
128
94
184
306
266
232
265
267
268
51
104
264
87
309
86
229
290
261
231
85
234
233
15
14
220
221
182
181
215
74
294
106
150
149
146
65
280
145
3
320
73
63
64
180
62
319
24
288
207
204
198
202
201
199
200
203
302
209
206
196
48
262
318
142
141
143
137
120
119
122
123
121
118
117
188
186
194
189
191
190
192
275
274
187
23
301
297
272
269
271
164
165
172
293
216
314
277
300
197
212
208
138
317
316
56
58
57
17
18
16
61
60
193
101
185
116
115
75
50
311
59
289
286
287
285
250
240
258
257
242
245
251
252
256
255
254
253
332
19
21
211
210
213
214
179
223
100
307
205
217
102
218
335
334
107
222
283
91
90
89
96
167
305
168
39
42
169
284
34
31
67
173
43
99
175
174
291
44
279
304
303
136
263
140
2
378
29
4
341
344
463
466
467
342
345
343
499
498
500
346
8
462
459
328
325
336
480
497
494
479
330
492
488
476
338
477
481
491
339
495
327
326
340
337
473
483
496
472
324
384
381
385
386
485
474
487
482
486
475
490
489
493
484
323
501
478
177
178
312
163
441
422
442
446
447
448
443
439
440
445
427
438
418
444
402
389
412
413
410
449
450
425
411
404
421
415
390
430
428
434
435
433
432
429
416
355
357
403
356
6
420
419
417
377
426
333
395
376
399
396
393
5
365
364
363
53
52
54
55
394
397
424
423
398
453
2
18
35
36
133
359
360
451
354
132
308
358
66
69
225
273
452
84
131
130
405
347
406
408
461
348
4
65
7
351
458
349
407
409
464
1
90
148
147
296
26
27
152
30
29
25
367
366
368
369
374
373
392
401
400
391
361
362
375
372
371
456
455
370
460
353
352
387
380
388
379
378
454
457
414
437
436
431
260
259
157
156
159
139
20
469
40
470
468
471
41
22
161
160
162
158
129
383
382
350
295
33
38
32
77
76
78
37
299
298
313
331
183
243
239
249
244
247
248
246
241
237
236
235
238
isolates
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
variable genes
Swine
52
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
53
Molecular adaptation
• Powerful statistical methods for detecting adaptive
molecular evolution
– Nonsynonymous substitution rate elevated above the
synonymous rate as evidence for positive selection
• Fixation of advantageous mutations, driven by natural
selection =>evolutionary innovations
• assess positive selection pressure across core
genome components of comparative taxa,
identifying genes and sites within genes of key
functional significance
54
Molecular adaptation analysis pipeline
Gene gain and loss
- pronounced genome flexibility in the
evolution of Campylobacter species
- many LGT islands, particularly C. coli
- large proportion of gained loci are
hypothetical genes
gene gain
gene loss
Positive selection
- large proportion of the core-genome under PS
- more PS on C. coli lineage than C. jejuni
- large proportion of PS loci are hypotheticals
Distribution of positive selection across lineages and genes
• high levels of PS in Campy
• Campy PS quite evenly distributed across
gene categories and rarely specific to a
lineage
• same loci frequently under positive
selection across multiple lineages
Evolution of Campylobacter diversity
Current and future activities…..
• Next generation genome sequencing [454 (collaboration with MSU) and
Solexa (Cornell)] of C. coli and C. jejuni isolates from different hosts and
putative ecotypes
– Solexa: comparative data across core genome of multiple strains
• New population genetic methods => genes under positive selection pressure
across clones and ecotypes of C. coli and C. jejuni;
• Identification of putative non-coding functional elements
– Phylogenetic footprinting
• Inter-specific genome wide alignments: conserved non-coding sequence elements
=> comprehensive picture of molecular adaptation in C. jejuni and C. coli =
genes of greatest functional significance to Campylobacter species and
ecotypes (e.g. what are the key molecular adaptation differences between
human and animal ecotypes?); evolutionary hypotheses that can serve as a
guide to mutation experiments, to assay functional significance.
FOOD & WATERBORNE DISEASES INTEGRATED
RESEARCH NETWORK
4.1.The role of the host microbiota in enteric
disease development
PD: Craig Altier
http://www.cvm.ncsu.edu/dphp/micro/altier.html
Co PDs: Bettina Wagner, Michael Stanhope, and Vincent Young
Project aims:
1.
2.
3.
Develop a mouse model to study the interaction of normal microbiota with the
intestine in enteric disease
Define a map of microbial population changes throughout the intestine that result
from antibiotic administration and lead to increased susceptibility to enteric
infection.
Characterization of protective innate and adaptive mucosal immune responses to
the Salmonella pathogen influenced by changes in the normal intestinal
microbiota.
60
A mouse model to characterize the intestinal
environment in normal and antibiotic-treated
animals
Characterization of the intestinal microbiota in
normal and antibiotic-treated mice
A.
OTUs
B.
OTUs
C.
OTUs
The ileum, the site of infection
by important enteric
pathogens, harbors a
microbiota distinct from other
areas of the intestinal tract.
The mucosal-associated
bacteria of this region also
differ from those of the
intestinal lumen, with
segmented filamentous
bacteria (SFB) comprising a
significant portion of the
population. Streptomycin
greatly changes this population
structure, reducing the
proportion of SFB present.
Cytokines/chemokines secreted by intestinal
tissues of mice infected with Salmonella +/streptomycin treatment
Intestinal tissues (ileum, colon, cecum) were cultured o/n and
cytokines/chemokines were measured in the supernatants using a
multipex assay and Luminex technology.
No detectable concentrations of IL-2, IL-4, IL-13 and TNF- in all
samples.
For FGF, IL-6, IL-10, IL-17, KC, MCP-1, VEGF no difference were
detected between both groups.
Significant differences between mice receiving Salmonella only and
Salmonella and with streptomycin
Cytokine/
chemokine
Ileum
Cecum
Cytokine/chemokine function
P<0.01
pro-inflammatory, Th1 cytokine,
promotes cellular immunity
P<0.05
growth factor, dendritic cell maturation
IL-1
P<0.001
pro-inflammatory
IL-1
P<0.05
pro-inflammatory
IFN-
GM-CSF
IL-5
P<0.001
eosinophil attraction and stimulation
P<0.01
IL-12
P<0.01
Th1 differentiation (IFN- induction)
IP-10
P<0.05
chemokine, IFN- inducible protein
MIP-1
P<0.01
chemotaxic factor for lymphocytes
and NK-cells
MIG
P<0.001
anti-bacterial, IFN- dependent
chemokine
Cytokines increased in streptomycin-treated mice are indicated with . Those
that were reduced in streptomycin-treated mice are indicated with .
The fatty acid environment of the intestinal
varies within specific regions of the tract
Cecum
Cecum
Ileum
Ileum
Intestinal fatty acids regulate Salmonella virulence,
with formate inducing expression of invasion genes,
and propionate and butyrate repressing it
FOOD & WATERBORNE DISEASES INTEGRATED
RESEARCH NETWORK
Summary
• Research focus areas at the Cornell ZRU
• Collaboration and associated projects
• Leveraging institutional resources
67
FOOD & WATERBORNE DISEASES INTEGRATED
RESEARCH NETWORK
Research Focus Areas at the Cornell ZRU
Network priorities (high priority areas in FWD IRN):
“Define the ecology and microbiology of food and waterborne zoonoses as well as drug-
resistant pathogens”
Project: Transmission of MDR Salmonella (ZC001-03)
Lorin Warnick and Martin Wiedmann
Project: Molecular Diagnosis of Bacterial Pathogens (ZC002-03) Yung-Fu Chang
Project: Sources and transmission of MDR Salmonella strains that cause human infection
(ZC006-07) Lorin Warnick
Statements of Objectives: “Antibiotic Resistance and Campylobacter”
Project: Molecular Evolution of Campylobacter Diversity (ZC003-05) Michael Stanhope
Statements of Objectives: “Intestinal Flora Research Areas”
Project: The Role of the Host Microbiota in Enteric Disease Development (ZC004-06) Craig Altier
Statements of Objectives: “Clostridium difficile”
Project: C. difficile: Comparative Genomics and Strain Differentiation (ZC005-06) Yung-Fu Chang
FOOD & WATERBORNE DISEASES INTEGRATED
RESEARCH NETWORK
Collaborations and Associated Projects
Washington State University
Sources and transmission of MDR Salmonella strains that cause human
infection (ZC006-07)
New York State Department of Health
Food Safety Research and Response Network
Johne’s Project
Michigan State University
The Role of the Host Microbiota in Enteric Disease Development
(ZC004-06)
Salmonella Enteritidis MLVA
Campy and Molecular Evolution
FOOD & WATERBORNE DISEASES INTEGRATED
RESEARCH NETWORK
Leveraging Institutional Resources
Infectious Disease Research Suite
PIs housed in new shared space
3000 square feet
Fully equipped for microbiology and molecular biology
East Campus Research Building
Recently opened state-of-the-art animal facility on Veterinary campus
Will allow continued and expanded opportunities for animal models of infectious
diseases
Animal Health Diagnostic Center Building
Will expand the current world-renowned animal diagnostic laboratory
Will provide biosafety level 3 space to the campus
Breaking ground in Spring of 2008, with anticipated occupancy in 2010
Cornell ZRU Abstracts
ZC001-03 Transmission of MDR Salmonella
Abstract: “The duration of fecal Salmonella shedding among dairy cattle following clinical disease”
ZC002-03 Molecular Diagnosis of Bacterial Pathogens
Abstract: “Microbial Diagnostic Array Workstation (MDAW): A Web Server for Diagnostic Array Data Storage,
Sharing and Analysis”
ZC003-05 Molecular Evolution of Campylobacter Diversity
Abstract: “Evolutionary genomic analysis of the genus Campylobacter”
Abstract: “A phylogenomic and strain genotyping examination of possible host adapted ecotypes in
Campylobacter coli”
ZC004-06 The role of host microbiota in enteric disease
Abstract: “Characterization of the Intestinal Environment in Conventional and Streptomycin-Treated Mice”
ZC005-06 C. difficile: Genomics/transciptome study
Abstract: “Comparative genomics of Clostridium difficile isolated from different hosts reveals genetic
differences relating to host species”
ZC006-07 Sources and transmission of multidrug-resistant Salmonella strains that cause human infection
Abstract: “Different Clones of Salmonella 4,5,12:I:- Isolates in Different Continents Causing Outbreaks”
Abstract: “Dynamics of infection in small transient populations”
Abstract: “The effect of clinical outbreaks of bovine salmonellosis on fecal shedding of MDR Salmonella”