Potential of plant-derived antimicrobials for controlling zoonotic and food-borne diseases
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Potential of plant-derived antimicrobials for controlling
zoonotic and food-borne diseases
Kumar Venkitanarayanan, DVM, MVSc, MS, Ph.D.Professor of MicrobiologyGraduate Programs Chair
Department of Animal ScienceUniversity of Connecticut
Storrs, CT 06269, USA
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About 75% of recently emerging infectious diseases affecting humans are diseases of animal origin, and approximately 60% of all human pathogens are zoonotic.
Food-borne diseases
Wide range of animal reservoirs
Emergence of antibiotic resistance
Zoonotic Diseases
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Phytophenolics
Plant defense mechanism
Wide spectrum of biological effects
Bacterial resistance low
Plant-Derived Antimicrobials (PDAs)
An alternative approach
Burt et al., 2004; Ohno et al., 2007; Wollenweber, 1988
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Plant-derived antimicrobials (PDAs)
• Trans-cinnamaldehyde (TC)
• Carvacrol (CR)• Thymol (TH)
• Eugenol –(EU)
• Caprylic acid –(CA)
•
Cinnamon oil
Oregano oil
Clove oil
Coconut oil
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Efficacy of PDAs for reducing egg-borne transmission of Salmonella Enteritidis
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• Major food-borne pathogen worldwide
• Highest incidence rate for Salmonella
infections1
• Total estimated annual cost – 4 billion USD2
1CDC 2010, 2Scharff, 2012
Salmonella
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Listeria monocytogenesNo reported outbreaks
SalmonellaPathogenic E. coli
Salmonella outbreaks in the US
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Source: CDC National Outbreak Reporting System, 2004–2008
Foods associated with Salmonella outbreaks
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• Chicken – reservoir host • Salmonella Enteritidis (SE) – most common Salmonella from poultry1
• Consumption of raw or undercooked eggs• 100 billion table eggs are produced annually
1 Marcus et al., 2007; 2 AMI, 2009; 3 CDC, 2010
Salmonella Epidemiology
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Primary colonization site – cecum
• Other sites – crop, intestine, cloaca
• Internal organs – liver, spleen, oviduct
Transmission routes:
• Horizontal – bird to bird
• Vertical – Transovarian - bird to yolk
(macrophages involved in systemic spread)1,2
1 Gast et al., 2007; 2 Gantois et al., 2009
Salmonella chicken link
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Controlling SalmonellaIdeal Intervention Strategy
• Economically viable
• Practical for farmers to adopt
• No toxicity
• Organic farming
• Environmentally friendly
• No bacterial resistance development
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Supplementation of PDAs
Reduce cecal colonization
Control salmonellosis
Reduce oviduct colonization
Reduce fecal contamination of
shelled eggs
Reduce yolk and membrane
contamination
Rationale
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13
• 120 White Leghorn layer chickens • 25 & 40 weeks of age
• Treatments
1% TC control (No SE, 1% TC)
1.5% TC control (No SE, 1.5% TC)
Positive control (SE, No TC)
1% TC (SE, 1% TC)
1.5% TC (SE, 1.5% TC)
SE-28, SE-21, SE-12, SE-31, SE-90
Challenge experiment in chickens
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1 2 10
Weeks
Day 1Groupingof birds
Day 10Challenge
withSE
Day 12Challengecheck
Day 14EggCollectionstarts
Daily egg collection
Weekly fecal swab
Day 70End of trial Collection
of tissues
TRANS-CINNAMALDEHYDE FEEDING PHASE
Protocol
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RESULTS
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Effect of TC on SE contamination of eggs *, week 1
25 WEEKS 40 WEEKS
ab b
a
b b
a
b b
a
b
c
*Treatments with different superscripts significantly differed from each other at P < 0.05
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Effect of TC on SE contamination of eggs *, week 2
25 WEEKS 40 WEEKS
a
b b
a
b
c
a
b b
a
b
c
*Treatments with different superscripts significantly differed from each other at P < 0.05
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Effect of TC on SE contamination of eggs *, week 3
25 WEEKS 40 WEEKS
a
b b
a
b
c
a
b b
a
b
c
*Treatments with different superscripts significantly differed from each other at P < 0.05
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Effect of TC on SE contamination of eggs *, week 4
25 WEEKS 40 WEEKS
a
b b
a
b
c
a
bc
a
b
c
*Treatments with different superscripts significantly differed from each other at P < 0.05
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Effect of TC on SE contamination of eggs *, week 5
25 WEEKS 40 WEEKS
a
b
b
a
b
c
a
b
c
a
b
c
*Treatments with different superscripts significantly differed from each other at P < 0.05
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Effect of TC on SE contamination of eggs *, week 6
25 WEEKS 40 WEEKS
a
bc
a
b
c
a
bc
a
b
c
*Treatments with different superscripts significantly differed from each other at P < 0.05
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Effect of TC on SE contamination of eggs *, week 7
25 WEEKS 40 WEEKS
a
b b
a
bc
a
cb
a
b c
*Treatments with different superscripts significantly differed from each other at P < 0.05
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Effect of TC on SE contamination of eggs *, week 8
25 WEEKS 40 WEEKS
a
b b
a
b b
a
b b
a
bc
*Treatments with different superscripts significantly differed from each other at P < 0.05
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Effect of TC on SE contamination of eggs (N=2195) cumulative data
No change in egg production due to TC supplementation
25 WEEKS 40 WEEKS
a
b b
a
b
c
a
b c
a
b
c
*Treatments with different superscripts significantly differed from each other at P < 0.05
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*p < 0.05
Effect of TC on SE in Chicken Organs*
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Impact of research
In-feed supplementation of TC could be used to reduce egg-borne transmission
of SE and improve microbiological safety of eggs.
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Varunkumar Bhattaram
PhD Student
Department of Animal Science
University of Connecticut
PI: Dr. Kumar Venkitanarayanan
Attenuation of Vibrio cholerae infection using plant molecules
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Vibrio cholerae (VC)
Causative agent of human cholera
Toxin-mediated watery diarrhea Life threatening dehydration and electrolyte
imbalance Extreme cases lead to kidney failure and death
Serogroups O1 and O139
Globally - excess of 300,000 cases; 7500 deaths
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Cholera: Route of Transmission
Feces
Contaminated Soil
Unsanitized Water
Food Human
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Cholera toxin
CTB Cholera toxin B subunit
CTA1 and CTA2 Cholera toxin A subunit
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Treatment/Control
Oral Rehydration Therapy
Fluids supplemented with electrolytes and salts
Antibiotics First antibiotic resistant strain – 1970 (Kitaoka et al., 2011) Multiple drug resistant Vibrio cholerae (Das et al., 2008;
Akoachere., 2013)
Vaccine
Oral vaccine – Not fully effective (WHO Guidelines, 2012)
Safe and Effective alternative strategy needed
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Effect of CR, TH and EG on cholera toxin production
Strains of Vibrio cholerae used in this study – VC 11623; BAA-25870 and BAA 2163
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Protocol for quantification of cholera toxin
V. cholerae with or without PDAS
Culture supernatant collected after 24
hours
Dilution
Cholera toxin custom kit(KPL
protein detector kit)
Std curve,& Quantification by
colorimetry
Purified cholera toxin B subunit Serial dilution
Fleming et al., 1988
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Effect of CR, TH and EG on cholera toxin production (VC 11623)
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Effect of CR, TH and EG on cholera toxin production (VC 569b)
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Effect of CR, TH and EG on cholera toxin production (VC 2163)
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Summary and Future Studies
PDAs could be potentially added in oral rehydrating
solution to control V. cholera infection in humans
Validate the in vitro results in a mammalian in vivo model
Characterize and delineate the mechanism of action CR, TH,
and EG
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Controlling aflatoxins in chicken feed using carvacrol and trans-
cinnamaldehyde as feed additives
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• Fungal toxins in feed ingredients• Aspergillus spp. - A. flavus, A. parasiticus• Routes of contamination of grains:
• Pre-harvest, post-harvest, and transportation • Processing of feed ingredients • Formulated feed after processing
Aflatoxins (AF) in feed
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Aflatoxin-contaminated feed
Consumption by Chickens
Impact on Chicken
performance Impact on
Human health
Aflatoxin residues in
chicken products
Bbosa et al., 2013; Herzallah et al., 2013
Economic losses to producersCarcinogenic and hepatotoxic effect
Why study AF?
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• Acute aflatoxicosis:• 50% mortality within 48 hours
• Chronic aflatoxicosis:• Decrease egg production• Decrease hatchability• Liver necrosis
Thrasher, 2012; Hamilton and Garlich, 1971
Poor body weight gain
Fatty liver syndrome
Aflatoxicosis in chickens
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Incubated at 25oC for 3 months
0, 1, 2, 3, 4, 8, 12 weeks
Aflatoxin concentration
200 g chicken feed
CR0%, 0.4%, 0.8%, 1.0%
TC0%, 0.4%, 0.8%, 1.0%
Farag et al., 1989; Razzaghi-Abyaneh et al., 2008
A. flavus NRRL 3357 or A. parasiticus NRRL 4123 or NRRL 2999 (5 log10 CFU/g)
Materials and Methods
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*Treatments differed significantly from the control (n = 6) (p<0.05)
Effect of CR on A. flavus aflatoxin production in chicken feed
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* All treatments differed significantly from the control (n = 6) (p<0.05)
Effect of CR on A. parasiticus aflatoxin production in chicken feed
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*Treatments differed significantly from the control (n = 6) (p<0.05)
Effect of TC on A. flavus aflatoxin production in chicken feed
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*Treatments differed significantly from the control (n = 6) (p<0.05)
Effect of TC on A. parasiticus aflatoxin production in chicken feed
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Protective effect of CR and TC on decreasing aflatoxin-induced cytotoxicity in hepatocytes
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AcuteAflatoxicosis
Aflatoxin consumption by chicken
Chronic Aflatoxicosis
Liver hemorrhage
Liver necrosis
Fatty liver
Doerr et al., 1983
Hepatotoxic effect of aflatoxins
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Human Hepatocytes (ATCC HepG2)
24 hours incubation to form monolayer in 96-wells plate
3 ppm or 10 ppm aflatoxin
Control0 %
CR0.05% & 0.1%
TC0.05% & 0.1%
Incubate for 18 hours at 37oC
Cytotoxicity % using LDH assay
Reddy et al., 2006; Zhou et al., 2006
Materials and Methods
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* All treatments differed significantly from the control (p < 0.05)
3 ppm10 ppm
Efficacy of CR in reducing aflatoxin-induced cytotoxicity in hepatocytes
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* All treatments differed significantly from the control (p < 0.05)
310 ppm 3 ppm
Efficacy of TC in reducing aflatoxin-induced cytotoxicity in hepatocytes
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• CR and TC reduced aflatoxins in chicken feed
• CR and TC did not change feed composition
• CR and TC significantly decreased aflatoxin-induced cytotoxicity in hepatocytes (p < 0.05)
Summary
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• Determine the stability of CR and TC in chicken feed during manufacturing process
• Field trials in chicken farms
Concluding remarks
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THANK YOU !
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