I Microbiological Risk Assessment: Meat and Poultry V Products R K.N. Bhilegaonkar

I Division Of Veterinary Public Health Indian Veterinary Research Institute Izatnagar ™ Introduction: Microbial food safety

™ Microbial Risk assessment steps with examples –Hazard identification –Hazard characterization –Exposure assessment –Risk characterization

™ Appropriate level of protection, Food safety Objective

™ Avian influenza Food Safety

¾ Increased global I trade ¾ Discerning and Important issue knowledgeable V consumer

R ¾ BSE & nv CJD: UK ¾ Melamine in milk/milk products: China I ¾ Pesticide residues in cold-drinks: India ¾ Avian Influenza: India and Asian countries Microbial Food Safety

™ Foodborne illness : Global magnitude difficult to I assess ƒ Deaths due to diarrhoeal diseases in 2005: 1.8 million ƒ Developed countries: 30% population V (INFOSAN WHO 2008) ƒ USA: 76 million cases of foodborne diseases ƒ 325,000 hospitalizations R ƒ 5,000 deaths ƒ India: Magnitude is unknown ƒ Diarrhoeal diseases (Food and waterborne) : I 18.6 million children under age of 5 ƒ 3,86,000 deaths in children (1 in every 5 global deaths in children) Transmission of Foodborne Diseases

Animal feed/environment/protozoans

I Food animals Manure

V Animal derived food products Plant derived products

R Food Processing Plants

I RTE Foods

Humans

Microbial Risk Assessment

• A powerful tool for management of food safety I • Properly designed MRA: Objective and systematic evaluation of information V • Helps risk manager to take informed decision on food safety issue • 1999: CCFH adopted principles and guidelines for R the conduct of MRA • Several Pathogen: commodity MRA : FAO & WHO, developed nations I – Salmonella in eggs – Listeria monocytogenes in cheese MRA: India

I • No systematic MRA studies : Meat and poultry products V • Authentic and exhaustive base line data not available R • Vast country: Diverse culture/ethnicity • Tremendous variations in eating habits and preferences I • Variations in risk patterns HazardHazard IdentificationIdentification I

Food Intaket/ Hazard Exposure V Characterization assessment MEAT & POULTRY R PRODUCTS I

Risk Characterization HazardHazard Identification I Identification ™ What are agents present in food? • Salmonella spp. V • Listeria monocytogenes • Aeromonas spp. R • STEC • Campylobacter spp. • Staph. aureus I • Rotavirus Food-agent relationship

Food-agent relationship! ™ Campylobacter Jejuni- Poultry ™ Listeria monocytogenes: Cheese/milk products ™ Clostridium perfringens: Meats ™ Aeromonas: Fish

™ No systematic surveillance studies in India ™ Several scattered reports: but gives fair idea ™ Prevalence in meat and poultry: 5 - 7% ™ RTE meats and poultry products: 0 - 3%

Western World: Important pathogen Indian context: Prevalence of non-typhoidal Salmonella less

™ 2541 serotypes ™ > 128 serotypes present in India ™ New added every year Salmonella from food of animal origin Agarwal (2009)

Type of food sample No. of No. positive (% ) positive samples examined Pork sausage 241 15 6.22 Cocktail sausage 208 9 4.32 Oxford sausage 33 1 3.03 Cooked ham 319 20 6.26 Ham garlic (salami) 346 18 5.20 Hot dog 137 3 2.18 Frankfurter 218 7 0.03 meat pie 75 8 10.66 Pork kebab 161 2 1.24 beacon 146 3 2.05 Luncheon meat 149 2 1.34 Fresh pork meat 132 12 9.09 Kofta 27 2 7.4 Total 2096 102 4.86 Foods Samples Samples Percent positive examined positive Goat 50 0 0 Liver Spleen 50 0 0 Lymph node 52 0 0 Fresh muscle 662 18 2.89 Total : 814 40 4.91

Foods Samples Samples Percent positive examines positive Sheep 80 7 8.75 Mutton, shammi kebab Mutton keema 21 1 4.76 Other products 18 0 0 Total : 119 8 6.72

Agarwal (2009) Foods Samples Samples Percent examined positive positive Poultry 76 7 9.21 Dressed frozen chicken Poultry meat 209 21 10.04 Chicken-N-ham 119 3 2.52 Chicken sausage 75 0 0 Chicken salami 81 0 0 Chicken kebab 68 3 4.41 Chicken 37 0 0 frankfurter Egg 319 2 0.62 Other products 42 2 4.76 Total : 1061 39 3.67

Agarwal (2009) Foods Samples Samples Percent examined positive positive

Milk/milk product 36 0 0 Raw milk

Dry milk powder 83 3 3.61

Milk chocolate 140 1 0.07 Khoya sweet 5 0 0 Cheese 51 0 0 Total : 315 4 0.12

Agarwal (2009) E. Coli Prevalence

™ Numerous reports ™ One of the most Important foodborne pathogen in Indian context ™ Meat and poultry products : 20-50%; up to 100% ™ Human diarrhoea ™ Animal diarrhoea

™STEC (EHEC): Isolations in recent years ™ Animals : Important reservoirs ™ Isolations from meats : Buffalo, sheep, goat, kebabs, sausages L.L. monocytogenesmonocytogenes

Source Prevalence Place Reference

Buffalo meat < 10% Gujarat Brahmbhatt and Anjaria (1993) Goat meat < 10% Bareilly Banurekha et al., (1998) Goat meat 6.66% Bareilly Barbuddhe et al., (2000) Sheep meat 7.4% Bareilly Barbuddhe et al., (2000 Various meats 3 - 8% Bombay Waskar (2005) produts Poultry meat 8.5% Nagpur Kalorey et al., (2005) Poultry : Most important transmitters I ™ Isolations from poultry meat: 20 - 50% up to 100% V ™ Other meats: 3 - 5% R I •Pathogen found in aquatic environment ¾Saline & brackish water ¾Drinking water ¾Treated & un-treated sewage ¾ Abattoir waste water ¾Colonize slow sand filters ¾ Fish – Major source

¾ Poultry, Mutton, beef, milk, etc. are also found to be contaminated Aeromonas: Prevalence in meats

Source Prevalence Place Reference

Mutton 24-37% Hisar Khurana and Kumar (1997) Poultry 32-38% Hisar Khurana and Kumar (1997) Poultry meat 16% Bareilly Kumar (1998) Poultry meat 16% Bareilly Ghatak (2005)

Eggs 12-22% Bareilly Agarwal (1997) Kumar (1998) Goat meat 12% Bareilly Kumar (1998)

Various RTE meat 14% Bombay Waskar (2005) products Hazard I Characterization V Describes adverse effects of Estimates particular organism R dose –response Severity of illness relationship Morbidity I Fatalities

Scattered reports: No systematic studies on disease occurrence ™ Prevalence in human (non typhoid) : 1 - 5% ™ Prevalence in healthy carriers: 1 - 2% L.L. monocytogenesmonocytogenes : PrevalencePrevalence inin HumanHuman

Source Prevalence Place Reference

Abortion (150 patients) 14% Mumbai Krishna et al., (1966) Abortion still births 3% - Bhujwala et al., (1973) Abortion 3.3% Northern Kaur et al., India (2007) Abortion 10% Nagpur Kalorey (2008) Abortion 6% Goa Barbuddhe (2008) PrevalencePrevalence ofof STECSTEC inin HumanHuman

Source No. of STEC Place Reference E. coli Diarrhoeal 1338 9 New Delhi Pamchandran patients and Verghese (1987) HUS patients 25 19 New Delhi Kishore et al., (1992) Haemorrhagic 240 18 Bareilly Kapoor et al., enteritis (1995) Diarrhoea and 67 15 Bareilly Banerjee et al., UTI infection (2001) & Ratore (2000) I ™ Human diarrhoea: 10 - 15% V ™ Many asymptomatic carriers ™ Industrialized countries: Manifestations are severe R ™ In Asian countries: Symptoms are milder. I Role in GB syndrome : needs to be explored • Emerging pathogen of importance : Dairrhoea • Implicated in extra intestinal infections

Source Prevalence Place Reference

Diarrhoea 6.5% Chennai Komathi et al., (1998) Diarrhoea 8% Kolkata Chaterjee and Neogy (1972) Diarrhoea 0.2% Vellore Jesudasan and koshi (1990) Diarrhoea 6.5% Bareilly Ghatak (2005) • Major cause infant and children diarrhoea • 100,000 to 150,000 deaths in children (Broor et al., 2003) • Prevalence in diarrhoeal cases: 5-71% – In acute diarrhoea : 20-30% (Incidence in animal diarrhoea: 10 -50% ( Cattle, goats, sheep, pigs

• Source studies in relation to food: Lacking – Occurrence in food : difficult to study Environment: Food matrix L. monocytogenes: Salmonella: Zero Zero tolerance in 25 g tolerance in 25 g or or ml in USA, ml 0 –100 in EU/Canada Dose-response relationship

Pathogen: Host factor: Virulence susceptibility characteristics Immune status Food Matrix

• Type of food, properties of food, storage conditions affects growth and survival of pathogen

Salmonella ™ The pH optimum : around neutrality (6.8-7.5). ™ Above 9.0 and below 4.0 being bactericidal. ™ pH values > 10 Salmonella rapidly die. ™ Temperature for optimum growth is 35°C-37°C ranging between 5°C and 46°C. As low as 2°C : reported for S. Typhimurium. ™ Freezing arrests growth, but not bactericidal.

Listeria : Relatively sturdy to temperature and pH changes, survives various processes SalmonellaSalmonella CharacterizationCharacterization Singh, 2004

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3% S. Anatum 3% SalmonellaSalmonella serovarsserovars inin buffaloesbuffaloes

Rough Weltevreden 8% 20%

Typhimurium 8%

Stanley 2% Anatum

Saintpaul 45% 5%

Orion 3% Lagos 3% 4, 12:i:- 2% Dublin Bareilly 2% 2%

Singh, 2004 SalmonellaSalmonella serovarsserovars inin CattleCattle

Weltevreden 1% Adelaide Typhimurium var 1% copenhagan Rough 1% 9% Anatum 1% Berta 1% Typhimurium Chester 25% 6%

Drogana 3%

Saintpaul 1% Rostock 1% Paratyphi B 1% Muenchen 1% Dublin 29% Mbandaka 3, 10 : r : - 1% 4% Heidelberg HavanaEnteritidis 1% 3% 4% Singh, 2004 SalmonellaSalmonella serovarsserovars inin GoatsGoats

Rough Other 27 serovars 22% 24%

Bareilly 3% Dublin 4% Weltevreden 9% 28 : b : enx 5%

Virchow Infantis 4% 7% Paratyphi B Typhimurium 5% Saintpaul 11% Stanley 3% 3%

Singh, 2004 SalmonellaSalmonella serovarsserovars inin birdsbirds Singh, 2004

Bovismorbificans Rough 2% Chester 1% Other 34 serovars 1% Eastbourne 1% 12% Enteritidis Weltevreden 8% 1%

Virchow 14%

Typhimurium 11% Gallinarum Stanley 43% 2% Lille 2% Indiana 2% •Virchow and Typhimurium are more common than Enteritidis. E.E. colicoli ™ Different virulence types ¾ EPEC Frequently reported ¾ ETEC ¾ EIEC ¾ EAggEC ¾ EHEC Several isolations in recent times ¾ DAEC ¾ CDTEC ™ Majority STEC are non O 157: H 7 ™ O 157: H 7 : Rare ™ Goat Isolates lack EAE gene: Significance not elucidated Listeria monocytogenes

• Virulence depends on strain • Studies on strain/serotype: virulence are available • 1/2a, 4b serotypes: Implicated in > 90% outbreaks

• Animal Human : Evidence of Zoonoses Bovine- human reassortants Porcine –human reassortants Atypical strains AntibioticAntibiotic ResistanceResistance

Increased antibiotic resistance of foodborne pathogens

™ Health related issues associated with resistant bacteria ¾ Treatment failures ¾ Reduced therapeutic options ¾ Increased severity of symptoms ResistanceResistance profileprofile ofof SalmonellaSalmonella VirchowVirchow

100.00%

90.00%

80.00%

70.00%

60.00%

50.00%

40.00%

30.00%

20.00%

10.00%

0.00% Ac Cs Ca Ci Do Tr Ak Ce T G Cf N S C K Fr

1986-1990 1991-1995 2001-2005

Cs: Cefoperazone; Ci: Ceftriaxone; Do: Doxycycline; Ce: cephotaxime; T: Tetracycline; Cf: Ciprofloxacin; N: Neomycin; K: Knamamycin; Fr: furazolidone N= 109 HostHost SusceptibilitySusceptibility

• Exposed population status –Normal adult –Infant – Elderly – Immuno-suppressed/diseased • Severity of disease and dose required will vary Food Intake/ Exposure Assessment

To consider appropriate food to include in model

Eg: Listeria monocytogenes:

Not linked Low risk

Bread, Cookies, cakes, soft drinks Meat/sea foods Likely risk

Cheese/ milk products Salmonella

™ Eggs, poultry meat, pork and other meats and products I are the common vehicles ™ Improperly pasteurized fluid milk, ice-cream cheese V and other milk products. ™ Unpasteurized orange juice, uncooked tomatoes, raw R alfa-alfa sprouts, etc. I Characterize range of pathways by which food is contaminated I Sources of Salmonella in foods ¾ Infected animals

V ¾ Animal excreta, cross-contamination in slaughter houses R ¾ Contaminated water ¾ Contaminated soil I ¾ Rodents, lizards and avifauna ¾ Insects Prevalence of Salmonellae in common house- pests/synanthropic animals. ` Agarwal (2009)

Sources No. examined No. positive (%) Cockroach 254 4 (1.57) I House-mouse 97 8 (8.24) Shrew* 122 12 (9.83) Fly 31 0 V Bat 48 3 (6.25) Ant 30 2 (6.66) R Wall-lizard 328 69 (21.03) Total : 910 98

*Triple infection of S. Anatum, S. Hvittingfoss and I S. Saintpaul in one shrew. Double infection of S. Saintpaul and S. Bareilly in one shrew and that of S. Typhimurium and S. Paratyphi-B in an another shrew. Prevalence of Salmonellae in amphibians Agarwal (2009)

I Sources No. examined No. positive (%) Snake* 187 16 (8.55) V Turtle 26 1 (3.84) Toad 733 78 (10.64) R I *Besides water snakes, these also include 20 cobra and other land snakes where form no Salmonella was isolated. Hazard Hazard Exposure identification ++ characterization ++ assessment

Mathematical models

Risk Characterization RiskRisk CharacterizationCharacterization

• Integration of Hazard identification, Hazard characterization and exposure assessment using mathematical models • To obtain Risk estimates: Qualitative and Quantitative • Severity of the adverse effects • Uncertainties associated with estimates (biological variations s/a differences in virulence, susceptibility of population, etc.) • Based on MRA set the public health goals and targets • Appropriate level of Protection (ALOP) is to be decided by authorities: Eg. Reducing incidence of Salmonellosis

from To 100 per 1,00,000 10 per 1,00,000 population population ALOP • Food Safety Objectives (FSO): The Maximum frequency and concentration of hazard in food at the time of consumption that provides ALOP to consumers

To Achieve ALOP of 10 per 1,00,000 Population of Salmonellosis

The incidence of Salmonellosis in a particular food should be brought down to 5% from 10% level

FSO Avian Influenza

Hazard I identification

V Hazard Risk R characterization Characterization Exposure I assessment Avian Influenza in India

¾¾ TremendousTremendous lossloss toto poultrypoultry IndustryIndustry I ™ Maharashtra and Gujarat 2006 ƒ 3.45 lakh birds culled V ƒ 6 lakh eggs & 53 MT feed destroyed ™ West Bengal and Tripura: 2008 R ƒ > 40 lakh birds culled ƒ 15 lakh eggs & 80,000 Kg feed destroyed I (10/32008, DAHD, Govt. India Media notification) ™ Assam, West Bengal (Darjeeling) and Sikkim: 2009: Exact estimates not available I 02/01/2009 Follow-up report No. 4 13/01/2009 Follow-up report No. 5 21/01/2009 Follow-up report No. 6 V 02/02/2009 Follow-up report No. 7 H5N1 19/02/2009 Follow-up report No. 8 04/03/2009 Follow-up report No. 9 R 16/03/2009 Follow-up report No. 10 I 30/03/2009 Follow-up report No. 11

Avian influenza in birds Receptor-Ligand for H5N1 I V Bird H5N1 R α 2,6 α 2,3 present in human deep in the lung Human Flu I viruses Phylogenetic analysis of NA gene of H5N1 viruses

100 G s/H K /ww26/ 00 E n vironm ent /H K /437- 10/ 99 T H A/ 1(K A N -1)/ 04 Ck /M Y S /5858/ 04 99 G s/C am bodi a/ 28/ 04 Ck /Laos /7 192/ 04 T H A /16/ 04 VT M Ck /V NM /27/ 03 99 Dk /V NM /40/ 04 lin e ag e V N M /1194/ 04 Ck /India/ 7964/ 06 Hat a y/04 100 Ck/ In d ia/ 7966/ 06 Ck/ In d ia/ 7972/ 06 Dk /G Y /497/ 06 F u jia n I Dk /Laos /3 295/ 06 lin e ag e 100 100 Z J/16/ 06 82 Dk /H N/ 856/ 06 Dk /F J/1734/ 05 Ck /Y ogjak art a /B B V et -I X /04 Ck /W ajo/ B B V M /0 5 Indo ne si an 97 ID N/ 5/ 05 lin e ag e Q a /S T /911/ 05 B H G s/Q H/ 65/ 05 B H G s/Q H/ 59/ 05 Ck/ M an ip u r/In d ia /59007/ 07 V 87 Ck/ M an ip u r/In d ia /59001/ 07 great c res ted grebe/ Denm ark /7498/ 06 Ck /S udan/ 1784-10/ 06 Ck /N igeria/ 641/ 06 Ck /C ri m ea/ 08/ 05 W hooping s w an/ M ongol ia/ 2 44/ 0 5 sw an/ S lov enia/ 760/ 06 Dk /E gy pt /2253 -3/ 0 6 G f/S T /1341/ 06 T y/S uz dal ka /1 2/ 05 Ck /A fghani stan/ 1207/ 06 R cygnus cygnus /Ira n/ 754/ 06 Ck/ In d ia/ 8292/ 06 Ck/ In d ia/ 8824/ 06 Ck/ In d ia/ 9254/ 06 Ck/ In d ia/ 8829/ 06 Ck/ In d ia/ 9253/ 06 Ck/ In d ia/ 13766/ 06 Ck/ In d ia/ 8361/ 06 Q ingh ai-like Ck/ In d ia/ 8362/ 06 lin e ag e Ck/ In d ia/ 7979/ 06 I Ck/ In d ia/ 9257/ 06 Ck/ In d ia/ 13767/ 06 Ck/ In d ia/ 18760/ 06 Ck/ In d ia/ 9386/ 06 Ck/ In d ia/ 15053/ 06 Ck/ In d ia/ 13777/ 06 Ck/ In d ia/ 9255/ 06 Ck/ In d ia/ 8299/ 06 Ck/ In d ia/ 13746/ 06 Ck/ In d ia/ 15339/ 06 0. 05 WHO Food safety information Avian Influenza

¾ Conventional cooking (> 700C) safe : Inactivate I virus ¾ In poultry meat: not killed by refrigeration or V freezing ¾ Home slaughtering and preparation is hazardous R ¾ Eggs contain virus both on outside and inside of shell ¾ No evidence of infection: If consumed properly I cooked poultry and eggs ¾ Greatest risk in handling and slaughter of live infected birds R I S Base line data on prevalence of K Foodborne pathogen C H Food Safety Pathogen characterization A R Objective A C Exact magnitude of human illness T E R Food intake/exposure assessment I ???? Z Food Safety A & T Human Health Source attribution ???? I O n