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 104/ml– Clinical: 108/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 subsp. paratuberculosis (MAP) paratuberculosis = Johne-disease (sheep, cow) 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: 103 – 104/ml• Number of bacteria producing enterotoxin, can

cause illness in humans: 106/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 – 104/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. 103-104/ml (Rarely cause illness)• Bacterial growth results in production of enterotoxin, and

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

• The emetic form: need 106-108/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: infected raw milk (sheep) and milk products made from raw milk (cheese, cottage cheese)

• 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 ↑, Cl- ↑, flakes, greyish-brown, putrid• Secondary contamination (most common) during milking,

handling• 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 Reservoir Source of contamination

Human disease

Enteropathogenic (EPEC)

Human Water Diarrhoea in infants (rare)

Enterotoxigenic (ETEC)

Human Water, foods, soft cheese (camembert, brie)

Acute, profuse, watery diarrhoea (traveller's diarrhoea) (occasionally)

Enteroinvasive (EIEC)

Human Salads, soft cheese 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: pasteurisation, low pH

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 infection3. Foot and mouth disease• Before symptoms (asymptomatic) milk can be

infected• Food poisoning/ food born illness: raw milk,

and cream

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

MRL values (μg/kg)

Milk Edible tissues

Benzylpenicillin 4 50

Cephalexin 100 200-1000

Danofloxacin 30 100-400

Oxytetracycline 100 100-600

Tilmicosin 40 50-1000

Moxidectin 40 50-500

Ivermectin - 30-100

Doramectin - 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-dichlorodiphenyltrichloroethane, 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 in concentrations above residue limit can not be detected

• Lipophylic in cream or butter the concentration can be higher

• Accumulate in mother’s milk higher (30 times) concentration and can be detected also today!

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

• * banned active substance

Compounds MRL values (mg/kg)

DDT* 0.04

Aldrin, dieldrin* 0.006

Hexachlorocyclohexane (HCH) isomers*

0.003-0.008

Endosulfan 0.004

Organic phosphoric acid esters

0.01

Pyrethrins, pyrethroids 0.02-0.05

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

• x: no criteria

Food /

product

Maximum concentration (mg/kg)

Cd Pb Hg As

Raw meat (beef, pork, lamb, poultry) 0.05 0.1 N.d. x N.d.

Edible offals (beef, pork, lamb, poultry) 0.5-1.0 0.5 N.d. N.d.

Fish 0.05-0.1 0.3 0.5-1.0 N.d.

Crustaceans, molluscs 0.5-1.0 0.5-1.5 0.5 N.d.

Game and products 0.1 0.5 0.5 1.0

Meat products 0.1 0.15 0.03 0.2

Milk N.d. 0.02 N.d. N.d.

Butter 0.02 0.02 0.02 0.1

Cheese 0.05 0.1 0.02 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 animal milk human

• Aflatoxins

• Ochratoxin

• Zearalenone

Mycotoxins

• Aflatoxin B1 and B2 in lactating cow by oxidation is transformed to 4-hydroxy-metabolites named „milk toxin” = aflatoxin M1 and M2

• 1-3% of the toxin of feed is excreted by milk

• The aflatoxin M1 can not be inactivated by pasteurisation and in fermented milk products is quite stable

• Genotoxic carcinogen effect of aflatoxin M1 is a little bit lower than that of B1

Mycotoxins

• Ochratoxin A (OTA) can be present potentially in the milk, but mainly in less toxic metabolite form (OTAα) – 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

Amino acid Bacterium

Histamine Histidine Lactobacillus spp.

(L. buchneri)

Tyramine Tyrosine Enterococcus spp.

(E. faecalis, E. faecium)

Lactobacillus spp. (L. brevis)

Phenyl-ethylamine

Phenylalanine 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 106 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 Radioactive isotope

Half-life Limit (Bq/kg)

Short and medium half-life

I-131

Cs-134

8 days

2.19 years

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 surfaces of plants/

roots dairy cattle• 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.