Milk hygiene aspects of zoonoses and non-zoonotic human pathogens

1
Milk hygiene aspects of zoonoses and non-zoonotic
human pathogens
2
Microorganisms in the milk

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

3
Contamination of raw milk and milk products by
pathogens

• 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)

4
Contamination of raw milk and milk products by
pathogens

• 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

5
Contamination of raw milk and milk products by
pathogens

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

6
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

7
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

8
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)

9
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)

10
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

11
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 8C (under 10C slow
  growing and no toxin production)
• !Only 10-30 of the S. aureus strains can produce
  enterotoxin (human strains 40-60)!

12
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

13
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)

14
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

15
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

16
Brucella spp.

• Resistance against heat or acid low (60Clt,
  pHlt4)
• 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)

17
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

18
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

19
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)
20
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

21
Yersinia enterocolitica

• Secondary contamination by faeces
• During milking, handling
• Prevention/treatment pasteurisation

22
Leptospira spp.

• Bovine leptospirosis - worldwide incidence
• Intravital infection ? mastitis
• Milk yellow, dense consistency
• At 4C raw milk viable for 2 month
• Prevention/treatment pasteurisation, low pH

23
Coxiella burnettii

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

24
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)

25
Viruses

• 1. Source of contamination human faeces, (poor
  personal hygiene) ? secondary contamination
• Prevention/treatment pasteurisation
• 2. intravital/primary infection

26
Viruses

• Tick-borne encephalitis
• Food poisoning/ food born illness raw milk,
  products made from raw milk (soft cheese)
• Rabies
• Can be detected in milk but no evidence of human
  infection
• Foot and mouth disease
• Before symptoms (asymptomatic) milk can be
  infected
• Food poisoning/ food born illness raw milk, and
  cream

27
Chemical contamination of milk
28
Chemical contaminants

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

29
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)

30
Most common substances contaminating milk

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

31
Milk hygienic importance of pharmacology residues

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

32
Milk hygienic importance of pharmacology residues

• 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

33
MRL values of milk and other edible tissues
Active substance MRL values (µg/kg) MRL values (µg/kg)
Active substance 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
34
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

35
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

36
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.)

37
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 mothers milk ? higher (30 times)
  concentration and can be detected also today!

38
Other important pesticides

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

39
The maximum residue limits (MRL) for milk and
milk products
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

• banned active substance

40
Contaminants of environmental origin

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

41
Contaminants of environmental origin - Toxic
heavy metals

• Cadmium (Cd), and lead (Pb)
• Poor absorption from intestinal tract (lt10)
• 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

42
Maximum metal content of food of animal origin
Food / product Maximum concentration (mg/kg) Maximum concentration (mg/kg) Maximum concentration (mg/kg) Maximum concentration (mg/kg)
Food / product 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

• x no criteria

43
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)

44
Mycotoxins

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

45
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

46
Mycotoxins

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

47
Biogenic amines

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

48
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)
49
Biogenic amines

1. 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.
2. Biogenic amines produced by bacteria can
   contaminated milk during cheese production (from
   instruments and equipment)
3. Bacteria of the starter cultures also can produce
   biogenic amines.

50
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.

51
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.

52
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

53
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

54
Potential effects of biogenic amines

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

55
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).

56
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)

57
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.

58
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

59
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 pHgt5.5 not available for
  plants (agricultural soils) but in forests the
  mushrooms can be considerably contaminated!

60
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.