understanding the risk of - meat science

Understanding the Risk of Salmonella Contamination in Beef!

Dayna M. Harhay, PhD!U.S. Meat Animal Research Center!

Clay Center, Nebraska!

What do we mean by Risk…?!

?

Salmonella and Foodborne Disease!

National Salmonella Surveillance Annual Summary 2009 3

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FIGURE 1

Laboratory-confi rmed Salmonella Isolates from human sources reported to CDC by age group and sex, 2009

27% of confirmed cases <1 – 4 yrs of age

CDC, Salmonella Annual Summary -‐ 2009

•  Salmonellosis – Non-‐typhoidal Salmonella enterica

•  > 2,500 serotypes (~1,700 noted for making animals sick)

•  U.S. cases per year:

–  Confirmed ~40,000 –  Es#mated ~ 1.4 million

Salmonella and Foodborne Disease!

h.p://www.ers.usda.gov/Data/FoodborneIllness/

National Salmonella Surveillance Annual Summary 2009 3

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FIGURE 1

Laboratory-confi rmed Salmonella Isolates from human sources reported to CDC by age group and sex, 2009

27% of confirmed cases <1 – 4 yrs of age

59% of deaths 65 – 80+ yrs of age

CDC, Salmonella Annual Summary -‐ 2009

•  Salmonellosis – Non-‐typhoidal Salmonella enterica •  > 2,500 serotypes (~1,700 noted for making animals sick) •  U.S. cases per year:

–  Confirmed ~40,000 –  Es#mated ~ 1.4 million

•  Generally self limiSng enteriSs but can be invasive –  HospitalizaSon ~15,000 per year –  Death ~1% of confirmed cases/yr

•  Human health cost ~ $330 million/yr –  Decreased producSvity –  Medical expense

•  Industry cost ~$100s of millions –  Product recall –  Plant closures and clean up –  Liability costs

Salmonella and Foodborne Disease

Fecal Bacteria ~1011 CFU/g feces 100 billion/g!

•  Primary habitat – animal large intesSne / feces –  “Hearty bug” also survives well in the environment

•  6 serotypes account for >50% human cases

20 serotypes account for >70% cases Are some serotypes more virulent than others?

•  Complex eSology ~10% cases aeributed to outbreaks (OB) ~90% sporadic

Many poten#al sources of Salmonella!

Complexity of Salmonella etiology!

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HHS Healthy People Ini#a#ve 2020 Salmonellosis Target: 6.8 cases / 100K people

Presently ~14 cases / 100K

Summary of 152 outbreaks represenSng 12,181 cases of illness – not all inclusive.

Sporadic Illness ~90%

OB ~10-‐15% of confirmed cases in U.S. aeributed to OB linked with contaminated meat consumpSon

M. Ellin Doyle et. al., (2009) hep://fri.wisc.edu/docs/pdf/FRI_Brief_Salmonella_Human_Illness_6_09.pdf

~40,000 confirmed cases / yr

hep://wwwn.cdc.gov/foodborneoutbreaks/

Salmonella in Ground Beef!Prevalence: ≈ 4.2%

Bosilevac et al., AEM 2009

Newport MDR-AmpC 2002 - 47 ill 2007 - 38 ill 2009 - 40 ill

Typhimurium ACSSuT 2002 - 59 ill 2009 - 14 ill

Newport MDR-AmpC 2009 – CA ~800K lbs

Typhimurium ACSSuT 2009 – CO ~466K lbs

Outbreaks:

Recalls:

Most commonly identified serotypes

Montevideo Anatum Muenster Mbandaka

21%!15%!

9%!6%!

Salmonella in Ground Beef!Prevalence: ≈ 4.2%



And yet there are no documented Montevideo outbreaks attributed

to ground beef…

Most commonly identified serotypes

21%!15%!

9%!6%!

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!"#~0.4% of outbreak cases

(1998-‐2011)

Sources of Salmonella in Beef!1)  External contamination: ➧ Contaminated trim ➧ Intervention failure

Brichta-‐Harhay et al., AEM 2008 74(20):6289-‐6297

89.6% (85.1 – 94.0%)

50.2% (40.9 – 59.5%)

0.8% (0.18 – 1.42%)

HTCT MulSple hurdle carcass processing intervenSons

Salmonella prevalence and levels at slaughter (n=3,040 carcasses)

Salmonella carcass contamination!

0 10 20 30 40 50 60 70 80 90

Sample no.

10-2

10-1

100

101

102

103

104

105

Enumeration

Pre-evisceration Carcass Positive

Log 1

0 CFU

/ 10

0 cm

2

10-2

10-1

100

101

102

103

104

105

0 10 20 30 40 50 60 70 80 90

Sample no.

Enumeration

Pre-evisceration Carcass Positive

Log 1

0 CFU

/ 10

0 cm

2 Hide EnumeraSon

Carcass EnumeraSon

Carcass Prevalence

  Salmonella contamination levels on cattle hides at harvest were found to vary!

  Carcass contamination was found to correlate with hide contamination!

Brichta-‐Harhay et al., 2008 AEM 74(20):6289-‐6297

EnumeraSon

EnumeraSon

Pre-‐evisceraSon carcass posiSve

Pre-‐evisceraSon carcass posiSve

Sample no.

Sample no.

Pre-‐harvest monitoring and interven#ons

Salmonella and MDR Salmonella on cattle hides and carcasses at slaughter (95% CI).!Hide: 89.6% (85.1 to 94.0%)

Pre-‐eviscera#on Carcass: 50.2% (40.9 to 59.5%)

Post-‐interven#on Carcass: 0.8% (0.18 to 1.42%)

MDR Salmonella: 16.7% (8.3 to 25.1%)

PS Salmonella: 72.9% (63.7 to 82.1)

MDR Salmonella: 11.7 (4.4 to 19.0%)

PS Salmonella: 38.5% (29.3 to 47.7)

MDR Salmonella: 0.33% (-‐0.03 to 0.70%)

PS Salmonella: 0.47% (-‐0.04 to 0.98)

PS Salmonella: Anatum

Muenchen Muenster Montevideo Mbandaka Meleagridis

Cerro Give

Kentucky Saint Paul

MDR Salmonella Newport

Typhimurium Uganda Agona Anatum Reading Dublin


Salmonella on Post-Intervention Carcasses!

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Noted for making both

humans and cattle sick.

Host adapted – causes

cattle enteric fever;

rarely causes disease in

humans.

Generally associated

with healthy cattle, but

if introduced into

ground beef from

infected cattle, could

they then be more likely

to cause disease in

humans?

Isolated from cattle lymph

nodes and/or from post-

intervention carcasses.

Isolated from ground

beef or identified in

ground beef outbreaks.

FIG. 7. A. Comparison of the serotypes most frequently isolated from clinically

infected cattle with those noted for frequently causing disease in humans and those

isolated from cattle lymph nodes, ground beef and post-intervention carcasses. B.

Graph depicting the distribution of Salmonella serotypes isolated from post-

intervention carcasses by sample day.

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$"#Post-Intervention Carcass Salmonella Serotypes Observed

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 320

2

4

6

8

10

Typhimurium - MDR

Onrike

Montevideo

Muenster

Reading - MDR

Anatum

Newport - MDR

Dublin - MDR

NT - MDR

Dublin

Sample Day

Nu

mb

er

of s

am

ple

s p

os

itiv

e

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te

ste

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ay

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Typhimurium DT104

Post-‐IntervenSon Carcass Salmonella Serotypes Observed

Dublin

Post

42.6%

57.6%

Are certain Salmonella better at surviving carcass processing interventions…?

PS Salmonella MDR Salmonella

n=24


Are some Salmonella better able to survive carcass processing interventions…?!

mRNA

Genome

Salmonella Transcriptomics: Direct sequencing of DNA that has been transcribed

 Genes (operons -‐ mRNA)  sRNA

Expose Salmonella to a given niche

Isolate mRNA

Convert mRNA to cDNA

Sequence Salmonella Transcriptome

Align sequence reads to annotated reference genome

Determine genes and sequences that are up or down regulated

with respect to control

Workflow:

Gene expression response of Newport MDR-AmpC to beef carcass interventions

Expose Salmonella to simulated multiple hurdle carcass interventions

108

104 - 103 102 - 101

2% Lactic acid 55°C pH ~3.5

80°C Carcass chill 4°C

Gene expression response of Newport MDR-AmpC to beef carcass interventions!

With these data we can learn how to target Salmonella intervenSon survivors and prevent their entry into final beef products.

Expose Salmonella to simulated multiple hurdle carcass interventions

108

104 - 103 102 - 101

2% Lactic acid 55°C pH ~3.5

80°C Carcass chill 4°C

761 (16%) genes showed expression:

  Heat shock

  Acid stress

  DNA repair

  Virulence response regulators

NCBA funded study: D.M. Brichta-‐Harhay, T.P. Smith & G.P. Harhay

Gene expression response of Newport MDR-AmpC to beef carcass interventions

10-1 100 101 102 103 10410-1 100 101 102 103 10410-1 100 101 102 103 10410-1 100 101 102 103 104 10-1 100 101 102 103 10410-1 100 101 102 103 104

FPKM Values clustered by KEGG pathway!

Treated! Untreated!

Pentose and glucuronate!interconversions!

Gamma-Hexachlorocyclo-!hexane degradation!

Peptidoglycan !biosynthesis!

Inositol phosphate !metabolism!

Glycerophospholipid !metabolism!

Glyoxylate / dicarboxylate !metabolism!

Methane metabolism!Riboflavin metabolism!

Lipoic acid metabolism!Folate biosynthesis!

Terpenoid backbone !biosynthesis!Bacterial chemotaxis!

Flagellar assembly!

Mismatch repair!

Sources of Salmonella in Beef!2) Internal contamination:

Fat trim containing lymph nodes that may harbor Salmonella.

Flank

Chuck

Cattle type Chuck Flank OverallCull Cow 1.0 3.9 2.5Fed Beef 0.3 1.0 0.7n=1,140 1.6

Percent Salmonella Prevalence

9 Serotypes observed from 18 posiSve nodes including: Cerro (6), Typhimurium (3), Montevideo (2), Newport (1), Anatum (1)

Prevalence of Salmonella in subiliac lymph nodes of cull and fed cattle at harvest!

Cull Beef from Region 3 slaughtered in Region 5

Fed Ca[le (n=1,501) Cull Ca[le (n=1,826)

3,327 LN tested – 266 posiSve for Salmonella (8%) The majority of LN (92%) were not found to be contaminated

Spring 20

10

Winter 20

11

Summer 20

110

10

20

30

40

50 Region 5Region 3

Sal

mo

nel

la p

reva

lenc

e (%

)

Spring 20

10

Winter 20

11

Summer 20

110

10

20

30

40

50 Region 5

Region 2Region 3

Sal

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nel

la p

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Salmonella serotypes isolated from caele lymph nodes

Fed Cattle LN Cull Cattle LN

Serotype % (n=266) (n=233) (n=33)Montevideo6 44.0 48.5 12.2Anatum 24.8 27.5 6.1Reading 4.9 5.1 3Thompson*13 3.8 3.8 3Meleagridis 3.0 3.4Kentucky 3.0 1.7 12.1C07 NT 2.3 2.6Mbandaka 2.3 1.3 9.1Muenchen*10 1.5 1.7Bredeney 1.1 9.1Infantis12 1.1 1.3Newport3 1.1 0.9 3Braenderup11 0.8 6.1Brandenburg 0.8 6.1Cerro 0.8 0.9Dublin 0.8 6.1Muenster 0.8 0.9Panama* 0.8 6.1Saint Paul9 0.8 0.4 3Cubana 0.4 3Give 0.4 3Kiambu* 0.4 3Typhimurium2 0.4 3Uganda 0.4 3

Total % 100 100 100

Prevalence: ≈ 4.2%


21%!15%!

9%!6%!


Gragg et al., 2012 FBPD submi.ed

Enumeration of Salmonella from fed cattle subiliac lymph nodes!

34%

No apparent serotype bias for lymph nodes containing higher levels of Salmonella

Serotypes observed: Montevideo Mbandaka Anatum InfanSs Muenster Muenchen Reading Thompson Meleagridis Kentucky CO7 NT

8% of total tested 15% of total tested

66%

*Limit of detecSon ~ 20 CFU/LN

*

618 LN tested, 144 posiSve (23%)

Gragg et al., 2012 FBPD submi.ed

Summary: 1° Salmonella Sources in Beef!

•  External - hide to carcass transfer in slaughter process!– Regional variation in serotypes and levels of

Salmonella observed!

•  Internal - fat trim containing contaminated peripheral lymph nodes!– Prevalence appears to be affected by region,

season, and animal type (fed > cull)!– Salmonella on hides may be entering peripheral

lymph nodes as the result of injury (bites or wounds) – Environmental component to the problem!

Salmonella prevalence on swine carcasses at harvest!Swine Processing – 2 plants in the Midwest:

Pre-‐Scald Pre-‐EvisceraSon

Final Carcass

91.2% 19.1% 3.7%

Predominant Salmonella serotypes observed: Derby Typhimurium Anatum London

MDR Salmonella Serotypes observed: Typhimurium Derby Agona

Schmidt et al., (2012) AEM 78(8):2716-‐2726

n=1520 n=1520

n=1520

Skin is not removed

Diversity of Salmonella isolated from swine carcasses!

Su 1 Su 2 F 1 F 2W

1W

2Sp 1 Sp 2

0

20

40

60

80

100

% S

alm

onel

la p

reva

lenc

e

A.

Derby

OtherUganda

Seftenberg

Bredeney

Johannesburg

Cerro

Brandenburg

Putten

Kentucky

Mbandaka

MontevideoMuenster

London

Anatum

Typhimurium

Su 1 Su 2 F 1 F 2W

1W

2Sp 1 Sp 2

0

20

40

60

80

100

% S

alm

onel

la p

reva

lenc

e

B.

OhioSaint Paul

Minnesota

Schwarzengrund

KrefeldInfantis

AgonaAnatum

Derby

Typhimurium Other

Season and Sample Day Season and Sample Day

Plant 1! Plant 2!

Pork pre-‐scald skin samples 95 samples per collecSon day

EnumeraSon of Salmonella on swine carcasses at harvest

Direct plaSng enumeraSon methods

HGMF

SPCM

0

50

100

150

200

% Prevalence% Enumerable

Sample point (n=1,520)% of enumerable

samples 0.1 1 10 100 1,000 10,000Prescald carcass 37.7 - - 279 176 106 12Preeviseration carcass 4.8 55 15 3 0 0 0Chilled final carcass 0.6 6 1 2 0 0 0

Frequency of enumeration (CFU/100cm2)

Prescald Carcass Prevalence and EnumeraSon

Schmidt et al., (2012) AEM 78(8):2716-‐2726

Summary - Salmonella in Pork !

•  Levels and serotypes found to vary by processing establishment!– Differences in point of origin or lairage?!

•  Direct plating Salmonella enumeration methods!– High throughput methods for assessing

differences in incoming levels!– Useful for determining intervention efficacy!

Final Thoughts…

Final Thoughts…

What we need is a tricorder!

Final Thoughts…

Reliable measures of pathogen prevalence in feedlot and dairy settings!

1) Where to target pre-harvest interventions!

2) When interventions are used can demonstrate that they worked!

Knowledge

Acknowledgments!US MARC Scien#sts and Technicians Terry Arthur Mick Bosilevac Norasak Kalchaynand John W. Schmidt Rong Wang Steven Schackelford Tommy Wheeler

Kim Kucera Julie Dyer Frank Reno Bruce Jasch Greg Smith

Former USMARC Mohammad Koohmaraie – IEH Michael N. Guerini

Texas Tech University Interna[onal Center for Food Industry Excellence Guy Loneragan Sara Gragg Mindy Brashears Chance Brooks Tyson Brown

Partners in Industry – Thank you!

Na#onal Ca[lemen’s Beef Associa#on Beef Industry Food Safety Consor#um (BIFSCo) Mandy Carr-‐Johnson Bo Reagan

Greg Harhay Tim Smith

Renee Godtel Bob Lee

USDA ARS FFSRU Tom Edrington

Na#onal Pork Board Steve Larsen

understanding the risk of - meat science

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