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 Cow, sheep,
(EHEC)
goat
Verotoxigenic (VTEC)
Raw meat, meat
Bloody diarrhoea
products, raw milk, milk (haemorrhagic colitis)
products
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 (Mouldripened 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 infection
3. 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 milkblood barrier from
the blood (lypophyl,
non-ionic, lessbinding 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
50
100
200-1000
30
100-400
100
100-600
40
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 (DDTdichlorodiphenyltrichloroethane, 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
Compounds
DDT*
Aldrin, dieldrin*
Hexachlorocyclohexane
(HCH) isomers*
Endosulfan
Organic phosphoric acid
esters
Pyrethrins, pyrethroids
•
* banned active substance
MRL values (mg/kg)
0.04
0.006
0.003-0.008
0.004
0.01
0.02-0.05
Contaminants of environmental
origin
1.
•
2.
•
Toxic heavy metals
Cadmium, lead, methyl-mercury
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
Maximum concentration
(mg/kg)
Cd
Pb
Hg
As
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
Raw meat (beef, pork, lamb, poultry)
•
x: no criteria
Contaminants of environmental origin
- Polychlorinated organic pollutants
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•
•
•
•
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)
Phenylethylamine
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 halflife
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.