Milk hygiene aspects of zoonoses and non-zoonotic human pathogens

Microorganisms in the milk

• Saprobes – 1, no illness (inhibit pathogens) – 2, no illness (organoleptic deviation – metabolism) • Pathogens – ILLNESS

Contamination of raw milk and milk products by pathogens

• •

1. Primary/Intravital infection

Systemic diseases (salmonellosis, tuberculosis, brucellosis, listeriosis, Q fever) → excretion by milk Mastitis – Subclinical: a few 10 4 /ml – Clinical: 10 8 /ml (pathogens! ↔ Somatic cell count)

Contamination of raw milk and milk products by pathogens

• • • •

2. Secondary (fecal) contamination

Most common During milking → hygiene deficiency of milking

Campylobacter

, EHEC,

L. monocytogenes

Other sources of contamination: air, dust, fixtures of stable, milking instruments, milk container, hand or clothes of stuff

Contamination of raw milk and milk products by pathogens

• • •

3. Recontamination (post-contamination)

After heat treatment Sources of contamination: stuff or environment (water, air, dust) and fixtures, instruments Especially dangerous (no way for killing)

Contamination of raw milk and milk products by pathogens

• In general: • Pathogens can cause intravital infection in animals (mastitis) → no in herds or very rare?

(tuberculosis, brucellosis; salmonellosis, listeriosis) • More common secondary contamination • WHO: 28 bacteria, viruses, unicellular pathogens can cause illness by milk consumption

Pathogens can contaminate raw milk

• Bacteria – Gram +: Mycobacterium spp.,

S. aureus

,

L. monocytogenes

,

Bacillus cereus

– Gram -: Brucella spp., Salmonella spp.,

Campylobacter

spp., EHEC,

Yersinia enterocolitica

, Leptospira spp.,

Coxiella burnetti, Streptobacillus moniliformis

• Viruses

Mycobacterium spp.

• •

M. bovis

,

M. tuberculosis

• Intravital/ primary infection (without clinical symptoms of mastitis)

Food poisoning/ food born illness

: Can live in fermented milk products made from raw milk (1-2 months)

Mycobacterium spp.

•

Food poisoning/ food-born illness

: Can live in fermented milk products made from raw milk • Pasteurisation: to prevent infection by

M. tuberculosis

. (phosphatase enzymes inactived – pasteurisation – mycobacteria are killed)

Mycobacterium spp.

•

Prevention/treatment

: pasteurisation and eradication from herds • Milk: only from herds officially free from tuberculosis and brucellosis (Reg. 853/2004/EC) – protection from infected humans •

Mycobacterium avium

(MAP)  (sheep, cow)  subsp.

paratuberculosis

paratuberculosis = Johne-disease secondary contamination by faeces • (Chron-disease) • MAP can survive pasteurisation

Staphylococcus aureus

• • Most common contagious pathogens (mastitis – from animal to animal)

Primary/ intravital infection

• Subclinical mastitis: 10 3 – 10 4 /ml • Number of bacteria producing enterotoxin, can cause illness in humans: 10 6 /ml • Milk have to be cooled ≤ 8°C (under 10°C – slow growing and no toxin production) • !Only 10-30% of the

S. aureus

strains can produce enterotoxin (human strains: 40-60%)!

Staphylococcus aureus

• • • Source of contamination: clothes, hands of staff  secondary contamination/ recontamination Prevention/treatment: S. aureus can be killed by pasteurisation but the toxins are thermostable

Food poisoning/ food born illness:

raw milk and fermented milk products made from raw milk

Listeria monocytogenes

• • Source of contamination: soil, gastrointestinal tract of animal 

secondary contamination

• 1-100/ml milk • (in sheep, goat  mastitis – 10 4 /ml)

Food poisoning/ food born illness

: cheese (soft) made from raw milk (e.g. Roquefort, camembert)

Bacillus cereus

• • • Source of contamination: soil • In raw milk max. 10 3 -10 4 /ml (Rarely cause illness) • • Bacterial growth results in production of

enterotoxi

n, and ingestion leads to two types of illness, diarrhoeal and emetic (vomiting) syndrome.

The emetic form

: need 10 6 -10 8 /g bacteria for toxin production in the milk (at this Nr  remarkable spoilage of milk, circumstances not appropriate)

The diarrhoeal type

: bacteria grow and produce toxin in the intestine but the vegetative form of bacteria are sensitive to acid in the stomach

Prevention/treatment:

Appropriate cool storage

Brucella spp.

•

B. melitensis

– Sheep, goat – Mediterranean area – In the EU more than 1000 case in the last few years •

B. abortus

(

B. melitensis

biovar. abortus) – Cow – Most of the European countries are officially free •

Food poisoning/ food born illness

raw milk (cheese, cottage cheese) : infected raw milk (sheep) and milk products made from • 100-100 000/ml

Brucella spp.

• Resistance against heat or acid: low (60°C<, pH<4) • Pasteurised milk: ø • Fermented milk products: ø • Pasteurised cream: ø • •

Butter made from raw cream: virulent for 30 days Soft and semi-hard cheese made from raw milk: +

• MILK: no organoleptic changes, somatic cell count (SCC) ↑ , Langhans-type giant cells • Milk: only from herds officially free from tuberculosis and brucellosis (Reg. 853/2004/EC)

Salmonella spp.

• Role of milk in Salmonella-induced food born illness is low • Rarely cause septicaemia and (as a consequence) mastitis (intravital infection) • Milk: SCC handling ↑ , Cl ↑ , flakes, greyish-brown, putrid • Secondary contamination (most common) during milking, •

Food poisoning/ food born illness:

• Pasteurised milk: ø • Fermented milk products: ø • Butter, soft and semi-hard cheese made from raw milk: viable for 30 days

Campylobacter spp.

• •

C. jejuni

,

C. coli

• (intravital infection) • Secondary contamination by faeces • Source of contamination: environment, milking equipments, hand or clothes of stuff • Thermophyl  Summer (longer storage without cooling)  food borne infection

Prevention/treatment

: cooling (inhibit growing) and pasteurisation

Enterohaemorrhagic

E. coli

(EHEC)

E. coli strain Enteropathogenic (EPEC) Reservoir

Human

Source of contamination

Water

Human disease

Diarrhoea in infants (rare)

Enterotoxigenic (ETEC) Enteroinvasive (EIEC)

Human Human Water, foods, soft cheese (camembert, brie) Salads, soft cheese Acute, profuse, watery diarrhoea (traveller's diarrhoea) (occasionally) Dysentery-like diarrhoea (mucous, blood) (occasionally)

Enterohaemorrhagic (EHEC) Verotoxigenic (VTEC)

Cow, sheep, goat Raw meat, meat products, raw milk, milk products Bloody diarrhoea (haemorrhagic colitis) and haemolytic uraemic syndrome (HUS) (occasionally)

Enterohaemorrhagic

E. coli

(EHEC)

• (Intravital infection – mastitis 0-2%) • Secondary contamination by faeces • Higher tolerance to acid  fermented milk products (virulent) • • • Infectious dose is low (10-100)

Food poisoning/ food born illness

: raw milk, products made from raw milk (Mould ripened soft cheeses)

Prevention/treatment

: pasteurisation

Yersinia enterocolitica

• • Secondary contamination by faeces • During milking, handling

Prevention/treatment

: pasteurisation

Leptospira spp.

• • Bovine leptospirosis - worldwide incidence • Intravital infection  mastitis • Milk: yellow, dense consistency • At 4°C raw milk: viable for 2 month

Prevention/treatment

pH : pasteurisation, low

Coxiella burnettii

• Worldwide common, resistant bacterium • Primary/intravital infection (no symptoms) • At 4°C raw milk: viable for months • Sensitive to low pH • Can survive flash and traditional pasteurisation at lower range (HTST – 72 °C, 15sec; LTLT – 62°C, 30min)

Rat-bite fever: Streptobacillus

moniliformis

• Rats are the main reservoir of the organism • Human infection is usually acquired either from a rat bite/scratch, handling infected rats, or, • In the case of the form of disease known as Haverhill fever, ingestion of milk or water contaminated with the organism (via rat urine)

Viruses

• • 1. Source of contamination: human faeces, (poor personal hygiene)  secondary contamination

Prevention/treatment

: pasteurisation • 2. intravital/primary infection

Viruses

• 1. Tick-borne encephalitis

Food poisoning/ food born illness

: raw milk, products made from raw milk (soft cheese) • 2. Rabies Can be detected in milk but no evidence of human infection • • 3. Foot and mouth disease Before symptoms (asymptomatic) milk can be infected

Food poisoning/ food born illness

and cream : raw milk,

Chemical contamination of milk

Chemical contaminants

• Intravital contamination – Treatment – pharmaceuticals – Environment • Secondary contamination – Environment – Milking, milk processing (e.g. additives)

Legislation

• Regulation 1881/2006/EC (mycotoxins, heavy metals, dioxin-like materials, Polycyclic Aromatic Hydrocarbons (PAH) • Regulation 2377/90/EEC (residue limits of pharmacologically active substances) • Regulation 2218/89/EEC (radioactive contamination of foodstuffs)

Most common substances contaminating milk

• Pharmaceuticals • Pesticides • Contaminants of environmental origin (heavy metals, polychlorinated organic materials) • Mycotoxins • Biogenic amines • Radioactive substances

Milk hygienic importance of pharmacology residues

• • 1. Treatment of mastitis Intramammal (direct) application Through the milk blood barrier from the blood (lypophyl, non-ionic, less binding to blood proteins)

Milk hygienic importance of pharmacology residues

• • • 2. Excretion by milk Some products/pharmaceuticals are not permitted to use for treatment of lactating cattle (e.g.: avermectins) For other products withdrawal time have to be determined (based on MRL value) MRL values of milk lower than of other edible tissues

MRL values of milk and other edible tissues

Active substance

Benzylpenicillin Cephalexin Danofloxacin Oxytetracycline Tilmicosin Moxidectin Ivermectin Doramectin

MRL values ( μg/kg) Milk Edible tissues

4 100 30 100 40 50 200-1000 100-400 100-600 50-1000 40 50-500 30-100 40-150

Milk hygienic importance of veterinary drug residues

• Unwholesome effects: Allergic reaction • 10 IU penicillin derivate (1 IU benzylpenicillin = 0.6 μg) • In the case of antibiotics, residues can inhibit starter cultures in the production of yoghurt, cheese and other fermented milk products • 0.01 IU/ml inhibit lactobacillus in fermentation

Detection of drug residues

• • Screening test: • inhibition method • Sample + bacterium suspension (

Bacillus stearothermophylus

var.

calidolactis

) + indicator  nutrient media • Growing: • Signs of metabolism: • Confirmative test: – ELISA-methods, – Chromatographic, – Spectroscopic, – Electrochemical analysis

Milk containing residue is NOT ACCEPTABLE for human consumption and for further processing and as feed for animals

Residues of pesticides

• Chemical pest control of plants • • Pest and insect control  Contaminated feed and drinking water • Excretion into milk • Typical characteristics/properties of pesticides occurring in milk: – Lipophyl – Easy absorption from intestinal tract – Poorly metabolised – Passive diffusion through the blood-milk barrier to milk • E.g.:

chlorinated hydrocarbons

, like DDT-group (DDT-

d

ichloro

d

iphenyl

t

richloroethane, dieldrin, aldrin, etc.)

Residues of pesticides

• In most countries of Europe were banned 40 years ago • Very persistent (Persistent Organic Pollutants – POPs) but now, the concentration in the environment is low • Lipophylic   in concentrations above residue limit can not be detected in cream or butter the concentration can be higher • Accumulate in mother’s milk today!

 higher (30 times) concentration and can be detected also

Other important pesticides

• Organic phosphoric acid esters • Pyrethrins, pyrethroids • Fast metabolisation  not present in milk in considerable concentration.

The maximum residue limits (MRL) for milk and milk products

Compounds MRL values (mg/kg)

DDT* Aldrin, dieldrin* Hexachlorocyclohexane (HCH) isomers* Endosulfan Organic phosphoric acid esters Pyrethrins, pyrethroids 0.04

0.006

0.003-0.008

0.004

0.01

0.02-0.05

• * banned active substance

Contaminants of environmental origin

• 1. Toxic heavy metals Cadmium, lead, methyl-mercury • 2. Polychlorinated organic pollutants Dioxins, furans, biphenyls

Contaminants of environmental origin - Toxic heavy metals

•

Cadmium (Cd), and lead (Pb)

• Poor absorption from intestinal tract (<10%) • Accumulation in the organism • Can be present in the milk • • Not considerable (EU: 3-5% of Cd taken by humans is from milk or milk products)

Methyl-mercury

• Good absorption from intestinal tract • Good passive transport through the blood-milk barrier  Can be present in the milk (at low conc.) • 10% of Hg taken by humans is from milk

Maximum metal content of food of animal origin

Food / product Raw meat (beef, pork, lamb, poultry) Edible offals (beef, pork, lamb, poultry) Fish Crustaceans, molluscs Game and products Meat products Milk Butter • Cheese x: no criteria Maximum concentration (mg/kg) Cd 0.05

Pb 0.1

Hg N.d. x As N.d.

0.5-1.0

0.05-0.1

0.5-1.0

0.1

0.1

N.d.

0.02

0.05

0.5

0.3

0.5-1.5

0.5

0.15

0.02

0.02

0.1

N.d.

0.5-1.0

0.5

0.5

0.03

N.d.

0.02

0.02

N.d.

N.d.

N.d.

1.0

0.2

N.d.

0.1

0.3

Contaminants of environmental origin - Polychlorinated organic pollutants •

Dioxins, furans, biphenyls

• Lipophyl • Persistent • Are disposed to accumulate in food chain • Can be present in food (among others milk, butter, cream) • Generally lower than the criteria (Reg. 1881/2006/EC)

Mycotoxins

• Contaminated feed  human animal  milk  • Aflatoxins • Ochratoxin • Zearalenone

Mycotoxins

• Aflatoxin B 1 and B is transformed to 4-hydroxy-metabolites named „milk toxin” = 2 in lactating cow by oxidation

aflatoxin M 1 and M 2

• 1-3% of the toxin of feed is excreted by milk • The aflatoxin M 1 can not be inactivated by pasteurisation and in fermented milk products is quite stable • Genotoxic carcinogen effect of aflatoxin M 1 little bit lower than that of B 1 is a

Mycotoxins

• Ochratoxin A (OTA) can be present potentially in the milk, but mainly in less toxic metabolite form (OTA α) – no criteria • Zearalenone (ZEN) can be present potentially in the milk, but mainly in metabolite form ( α and ß-ZEN) – no health risk for humans

Biogenic amines

• Biogenic amines are formed from amino acids by microbial decarboxylation • Low concentration in milk but higher in some cheese

Biogenic amines in cheeses

Biogenic amine

Histamine Tyramine Phenyl ethylamine

Amino acid Bacterium

Histidine

Lactobacillus

spp. (

L. buchneri

) Tyrosine Phenylalanine

Enterococcus

spp. (

E. faecalis, E. faecium

)

Lactobacillus

spp. (

L. brevis

)

Enterococcus

spp. (

E. faecalis, E. faecium

)

Biogenic amines

a) Bacteria can produce biogenic amines (decarboxylase enzyme). They can be found in raw milk in higher amount → The amount of biogenic amines is higher in cheese produced from raw milk.

b) Biogenic amines produced by bacteria can contaminated milk during cheese production (from instruments and equipment) c) Bacteria of the starter cultures also can produce biogenic amines.

Biogenic amines

• • The required number of bacteria is 10 6 cell/g cheese

Enterobacteria

can not proliferate to reach this number in case of hygienic production technology →

Lactobacillus

and

Enterococcus

species are the main biogenic amine producers.

Biogenic amines

Prevention

: • Pasteurisation • Appropriate milking hygiene and handling of raw milk (cheese made from raw milk) • Use of proper starter culture • Efficient cleaning and disinfection of rooms, instruments and equipment.

Potential effects of biogenic amines

• Generally not dangerous for consumer (decomposed in the intestinal tract), but • Large amount uptake at once or • Not appropriate detoxication mechanism (gastrointestinal diseases, genetic defect) • TOXICATION

Potential effects of biogenic amines

•

Symptoms

(depends on the generating agent): • Histamine: – Drop in blood pressure – Dyspnoea – Urticaria – Nausea – Diarrhoea • Tyramine, phenyl-ethylamine: – Strong headache – Vomiting – Tachycardia – Increase in blood pressure – Visual disturbance

Potential effects of biogenic amines

• Toxic level of biogenic amines in food: • > 400mg/kg histamine • > 100mg/kg tyramine • > 30mg/kg phenyl-ethylamine • Related legislation (criteria): Reg. 2073/2005/EC (only fish! No cheese!)

Radioactive substances

• Radioactivity has always been a part of our environment. The substances in the world around us all contain

unstable atoms

that emit energy as radiation when they decay to more stable atoms. Such radioactive atoms are also called radionuclides.

• The air that we breathe, the water we drink, the food we eat, and our own bodies all contain radionuclides (K-40, Bi-214, Tl-205).

Radioactive substances

• The air, the soil, the water, the animal and the plant can be contaminated with artificial radioactive isotopes by atmospheric nuclear tests or nuclear reactor accidents.

• Biological effects depend on: – Radiation type and energy – Half-life – Interaction with biological tissues (in which accumulated – e.g. I-125, I-131 in thyroid or Sr-90 in bones)

Radioactive isotopes potentially present in milk

Group

Short and medium half life

Radioactive isotope

I-131 Cs-134

Half-life

8 days 2.19 years

Limit (Bq/kg)

500 1000 Long and ultra long half-life Sr-90 Cs-137 28 years 30 years 125 1000 • The becquerel (symbol Bq) is the SI derived unit of radioactivity. One Bq is defined as the activity of a quantity of radioactive material in which one nucleus decays per second.

Artificial radionuclides

•

I-131

• Accumulation in the udder • Significant amount excrete with milk • Half-life: 8 days (short)  • Acute contamination and damaging • Dysfunction and morphological changes in thyroid

Artificial radionuclides

• Cs-134,

Cs-137

• Half-life: 2 years,

30 years

• Radioactive cloud/ dust  roots  dairy cattle surfaces of plants/ • Good absorption from gastrointestinal tract • K + -like behaviour in the organism • Excreted also into milk • Strong binding to soils pH>5.5 not available for plants (agricultural soils) but in forests the mushrooms can be considerably contaminated!

Artificial radionuclides

•

Sr-90

• Half-life: 28 years • Ca ++ -like behaviour in the organism  accumulation in bones  damage the bone marrow, leukaemia • Mainly in the milk and milk products can be found in considerable concentration.