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PRION PROTEIN IN MILK

flounder

Well-known member
Subject: Prion Protein in Milk
Date: January 14, 2007 at 5:41 pm PST
PLoS ONE. 2006; 1(1): e71.
Published online 2006 December 20. doi: 10.1371/journal.pone.0000071.
Copyright Franscini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Prion Protein in Milk

Nicola Franscini,1 Ahmed El Gedaily,1 Ulrich Matthey,1 Susanne Franitza,1 Man-Sun Sy,2 Alexander Bürkle,3 Martin Groschup,4 Ueli Braun,5 and Ralph Zahn1*

1Alicon AG, Schlieren, Switzerland
2Institute of Pathology, Biomedical Research Building, Case Western University School of Medicine, Cleveland, Ohio, United States of America
3Lehrstuhl Molekulare Toxikologie, University of Konstanz, Konstanz, Germany
4Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Greifswald, Gemany
5Departement für Nutztiere, University of Zurich, Zurich, Switzerland
Matthew Baylis, Academic Editor

University of Liverpool, United Kingdom



* To whom correspondence should be addressed. E-mail: [email protected]
Conceived and designed the experiments: ZR FN EA MU FS RZ NF AE UM SF. Performed the experiments: FN EA MU FS NF AE UM SF. Analyzed the data: ZR FN EA MU FS RZ NF AE UM SF. Contributed reagents/materials/analysis tools: AB MG SM BA GM BU MS UB. Wrote the paper: AB MG ZR FN BA GM BU RZ NF UB.


Received October 19, 2006; Accepted November 6, 2006.


ABSTRACT


Background
Prions are known to cause transmissible spongiform encephalopathies (TSE) after accumulation in the central nervous system. There is increasing evidence that prions are also present in body fluids and that prion infection by blood transmission is possible. The low concentration of the proteinaceous agent in body fluids and its long incubation time complicate epidemiologic analysis and estimation of spreading and thus the risk of human infection. This situation is particularly unsatisfactory for food and pharmaceutical industries, given the lack of sensitive tools for monitoring the infectious agent.


Methodology/Principal Findings
We have developed an adsorption matrix, Alicon PrioTrap®, which binds with high affinity and specificity to prion proteins. Thus we were able to identify prion protein (PrPC)–the precursor of prions (PrPSc)–in milk from humans, cows, sheep, and goats. The absolute amount of PrPC differs between the species (from µg/l range in sheep to ng/l range in human milk). PrPC is also found in homogenised and pasteurised off-the-shelf milk, and even ultrahigh temperature treatment only partially diminishes endogenous PrPC concentration.


Conclusions/Significance
In view of a recent study showing evidence of prion replication occurring in the mammary gland of scrapie infected sheep suffering from mastitis, the appearance of PrPC in milk implies the possibility that milk of TSE-infected animals serves as source for PrPSc.


INTRODUCTION


Prion protein was detected in attempts to identify the infective agent of TSE [1], [2]. The finding that prion protein is present in normal and TSE-infected brain at similar levels [3], [4] suggests that the “cellular” prion protein (PrPC) constitutes a precursor of the “scrapie” prion protein (PrPSc) causing TSE such as bovine spongiform encephalopathy (BSE) in cattle or Creutzfeldt-Jakob disease (CJD) in humans. There is convincing evidence that the transition from precursor protein to infectious prion is due to a major conformational transition [5].

Prion protein is highly conserved among mammals [6]. It is primarily synthesized in cells of the central nervous system [7], but is also abundantly expressed in several peripheral tissues [8], [9]. An amino-terminal signal sequence targets prion protein to the endoplasmatic reticulum, where it transits the Golgi and ultimately reaches the external surface of the cell membrane [10]. There it is attached to a carboxy-terminal glycosyl phosphatidylinositol anchor [11]. The mature bovine protein of 217 amino acids contains two consensus acceptor sites for addition of N-linked polysaccharides [12].

Prion proteins (PrPC and PrPSc) have been detected in the cellular fraction of blood [13]–[17], but so far not in milk [18]–[21]. Considering that milk and milk products represent a major component of human nutrition it seems of particular importance to analyze milk for the presence of prion proteins. A first step in this direction is to determine the amount of PrPC in milk of healthy animals. If milk contains a significant amount of PrPC, this could indicate that PrPSc might be present in so far undetectable amounts in milk of TSE infected animals. However, the high concentration of total protein (about 40 mg/ml) and the high amount of lipids (about 35 mg/ml) in the milk make prion protein analysis by common biochemical methods demanding. We have therefore developed an adsorption matrix, Alicon PrioTrap®, which binds with high affinity and specificity to prion proteins PrPC and PrPSc. The exceptional binding properties of Alicon PrioTrap® result from hydrophilic and hydrophobic surface clusters that recognize different prion protein epitopes, allowing quantitative enrichment of extreme low quantity of prion proteins in body fluids and in biological tissues.


RESULTS


The detection of native PrPC after enrichment from 10 ml milk from cow, sheep, goat, and human using Alicon PrioTrap® is shown in figure 1. In cow milk three PrPC isoforms are observed with an apparent molecular mass of about 34 kD, 30 kD, and 27 kD corresponding to diglycosylated, monoglycosylated, and unglycosylated PrPC, respectively. In some preparations monoglycosylated PrPC appears as a double band, indicating that the two glycosylation sites may be linked to different carbohydrates. The apparent molecular mass of unglycosylated PrPC is slightly higher when compared to a recombinant bovine PrP(25–241) standard at 26 kD, indicating that native PrPC in milk contains a glycosyl phosphatidylinositol anchor [11]. About the same distribution of PrPC isoforms is observed for sheep, goat, and human milk, although the total amount of native PrPC significantly differs between the species. The relative ratio of sheep/cow/goat/human PrPC is estimated at 100/20/4/1. From experiments performed on sequential incubations with Alicon PrioTrap® the total concentration of PrPC in fresh cow milk can be estimated to be about 200 pg/ml. Taking into account the relative ratios of PrPC in milk of different species, fresh sheep milk and goat milk contain about 1 ng/ml and 40 pg/ml PrP, respectively. Human breast milk contains less than 10 pg/ml PrPC. The concentration of PrPC in Swiss off-the-shelf milk is reduced when compared to fresh milk, but prion protein can clearly be detected (Figure 1). About the same concentration of PrPC was measured for organic farm milk and non-organic farm milk as well as for pasteurized and ultra-high temperature (UHT) treated milk (data not shown).

To confirm specificity of immunochemical detection of PrPC in milk, we compared different anti-PrP monoclonal antibodies, which are directed against non-overlapping epitopes (Figure 2): PrP−mab 8B4 binds to residues 37−44 of mouse PrP [22]; mAB 6H4 targets residues 144−152 [23]; and PrP−mab 8H4 binds to residues 175−185 [24]. The three antibodies recognize the same proteins and thus confirm the presence of PrPC in milk. In control experiments, with non-PrP antibodies, e.g., anti-Tau protein monoclonal antibody (Chemicon International) (Figure 2) and anti-Aβ monoclonal antibody (Calbiochem, Germany; data not shown), none of the PrPC isoforms was detected, thus confirming binding specificity of the anti-PrP monoclonal antibodies. An interesting observation with regard to antibody 8B4 is its “clear” detection profile when compared to 6H4 and 8H4 antibodies. This can be rationalized by 8B4 not recognizing a variety of carboxy-terminal fragments of milk PrPC, which appear as smear in the Western Blot.

We further compared the glycoforms of native prion protein in cow milk with those of bovine brain, a tissue where prion protein expression is well characterized. The glycoforms were identified by digestion with PNGase (Figure 3), an enzyme that cuts off oligosaccharides from N-linked glycoproteins, e.g., the two N-linked sugars of PrPC [12]. After partial cleavage with PNGase the upper PrP-isoform in the Western Blot representing diglycolysated PrPC (34 kD) disappears in favour of monoglycosylated (30 kD) and nonglycosylated PrPC (27 kD). In parallel, there seems to be a small shift from the higher molecular weight monoglycosylated form to the lower molecular weight form. A slight downshift of the monoglycosylated PrPC is also observed for brain homogenate after PNGase treatment (Figure 3). The diglycosylated PrPC isoforms differ slightly in molecular mass, indicating that carbohydrate structure of PrPC in milk and brain may not be identical. More stringent reaction conditions result in complete truncation of carbohydrates from PrPC. Most importantly, the apparent molecular masses of nonglycosylated PrPC in milk exactly matches with that of the corresponding PrPC in brain homogenate.

Alicon PrioTrap® can also be applied for elimination of prion protein from milk. As shown in Figure 4, after the first treatment of 10 ml milk with Alicon PrioTrap® more than 95% of endogenous PrPC was already removed, and after the second treatment PrPC was completely eliminated. However, the overall protein concentration (measured by bicinchoninic acid assay, Pierce) was constant with about 40 mg/ml before and after PrPC elimination. The protein composition of milk as analyzed by SDS PAGE (Figure 4) was not affected either. Prion protein was also completely removed, when milk was spiked with PrPSc from mouse Rocky Mountain Laboratory (RML) brain homogenate (data not shown). Thus, Alicon PrioTrap® can be used for enrichment and detection of overall prion protein in milk, but also for complete removal of prions.


DISCUSSION


Milk contributes 13% to the worldwide protein supply for humans. World milk production ranges around 500 million tons a year. Before fresh milk reaches the consumer, it is usually homogenized to reduce fat particle size in order to increase digestibility of the milk and heated. Surprisingly, pasteurisation (heating for 30 seconds to 72°C) and ultra-high temperature treatment (heating for 1–4 seconds to 135°C) only leads to a partial reduction of the amount of PrPC. This supports the observation that PrPC is highly stable in milk. Thus, the heating procedures used to inactivate DNA-containing pathogens are not sufficient to eliminate endogenous prion proteins.

The presence of PrPC in blood has been documented [14], [15], and is confirmed by our own unpublished observations. To produce one liter of milk, about 400 to 500 liters of blood must pass through the udder of a cow. It is thus possible that the PrPC found in milk derives from blood cells or, alternatively, has been secreted from glandular epithelial cells. Cell types that have been identified in milk from healthy cows are mainly macrophages, and other leucocytes. However, in our assay cells are completely removed by centrifugation. Therefore, the recovered PrPC is not cell associated but most likely binds to other proteins or lipids resulting in stable molecular complexes. The fact that milk contains full-length PrPC, very likely comprising the glycolipid anchor, indicates that prion protein was originally cell-bound and does not represent any of the amino-terminal truncation products of PrPC known to be released from normal cells under physiological conditions [25]. The detection of such a considerable expression of cell membrane bound or derived PrPC in milk constitutes one of the key requirements for the generation of infectious prions in the udder of infected animals.

Over the last 10 years, scientific groups, risk assessment agencies, and public health organizations have debated the TSE risk for milk and milk products [26], [27]. Epidemiological and bioassay data so far available have not provided evidence for milk to harbour prion infectivity and infectious prions have as yet not been detected by bioassays in the milk, colostrum or udder of clinical BSE cases in cow [18]–[21]. However, a recent statement of the European Food Safety Authority affirmed that based on a number of observations from research data, there are indications that infectivity in the milk from small ruminants can not be totally excluded [28]. Furthermore, the exclusion of animals with mastitis, an inflammation of the mammal gland, being able to destabilize the blood-milk barrier, is considered a measure able to reduce but not to eliminate the potential contamination risk [28]. The rational of this conclusion is confirmed by a recent study showing that in sheep naturally affected with both scrapie and lymphocyte or lymphofollicular mastitis, PrPSc accumulation was present in lymphoid follicles adjacent to milk ducts [29]. At least in natural sheep scrapie, prion replication can occur following a lymphotropic virus infection in the inflamed mammary gland. This study has not detected PrPSc or prion infectivity in milk itself. However, since under such inflammatory conditions, the total number of immune cells increases in milk of animals, it might be possible that infectious PrPSc is also passing through and reaches the milk. In this context milk from such animals could possibly be responsible for the spread of scrapie from the ewes to their offspring in affected sheep or goat flocks. Moreover, sheep and goat milk could also constitute a TSE exposure risk for mammals (humans) consuming these products.

The former Scientific Steering committee of the European Commission and the European Food Safety Authority recommend that research should intensify on the safety of milk of small ruminants with regard to TSE risk. Limited new data are expected to be published in the near future and there is still little research initiated in this area [28]. The Alicon PrioTrap® technology opens a new avenue for studying the biochemical characteristics of prion protein in milk and thus may contribute to offer a feasible approach to perform an appropriate study on the milk safety with regard to TSE risk.


MATERIALS AND METHODS


Preparation of milk samples
Fresh milk was obtained from healthy individuals and transported at 4°C. Cow UHT milk, sheep and goat pasteurised milk were obtained from the Swiss market. Each sample was prepared from 10 ml milk, centrifuged at 3000× g for 10 min to ensure complete removal of cells.


Preparation of brain homogenate
10% (w/v) bovine brain homogenate was prepared in 100 mM Tris-HCl pH 7.5 containing 2% sodium lauryl sarcosinate. This solution was diluted in 100 mM sodium phosphate buffer pH 8 containing 0.5% NP-40 to obtain 1% (w/v) brain homogenate.


Concentration of milk PrPC
The milk supernatant was stirred for 30 min in the presence of Alicon PrioTrap®. The resin was centrifuged for 2 min at 2000× g and washed three times at RT with 10 ml washing solution containing 100 mM sodium phosphate buffer pH 8.


Immunochemical PrPC detection
Concentrated milk and brain PrPC was denaturated in SDS sample buffer and heated at 70°C for 10 min and at 95°C for 5 min, respectively. Samples were applied to a 12% SDS polyacrylamide gel for electrophoresis and subsequently transferred to a PVDF membrane. The membrane was blocked with 2% ECL Advance™ blocking agent (Amersham) if probed with 8B4 antibody or 1% bovine serum albumin if probed with other antibodies used in this study. 8B4 antibody was incubated at a concentration of 450 ng/ml, 8H4 at 85 ng/ml, 6H4 at 285 ng/ml, and anti-Tau-1 at 50 ng/ml. Sheep anti-mouse IgG horseradish peroxidase-conjugated secondary antibody (Amersham) was incubated at a 1/20,000 dilution. The immunoreactivity was visualized by chemiluminesecence detection following the manufacture's instruction (ECL Advance Western Blot detection Kit, Amersham).


PNGase treatment of milk and brain PrPC
For PNGase treatment prion protein extracted from 10 ml cow milk or 10 µl of 1% (w/v) cow brain homogenate was incubated for 12 h at 37°C in buffer containing 100 mM sodium phosphate, 10 mM Tris-HCl, 1% NP-40, 1% MEGA-8, pH 8, and 1.5 units of N-Glycosidase F (Roche). Under more stringent cleavage conditions, proteins were denatured by heating for 10 minutes at 100°C in the presence of 0.5% SDS before treatment with 4 units of N-Glycosidase F.


Total milk protein detection
A milk volume corresponding to 40 µg total protein (1 µl) was heated in SDS loading buffer at 70°C for 10 min. After electrophoresis on a 12% SDS polyacrylamide gel, the proteins were detected by silver staining (SilverSNAP Stain kit II, Pierce).


Acknowledgments

We thank Theo Bearth, Roland Ermini, Peter May, Günter Kreissel, and Mathias Schmid for their generous support. We are grateful to Torsten Seuberlich from the NeuroCenter of the University of Bern for kindly providing us brain homogenate from cattle.


Footnotes

Competing Interests: The authors have declared that no competing interests exist.
Funding: The authors have no support or funding to report.




References
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15.Barclay GR, Houston EF, Halliday SI, Farquhar CF, Turner ML. Comparative analysis of normal prion protein expression in human, rodent, and ruminant blood cells by using a panel of prion antibodies. Transfusion. 2002;42:517–526. [PubMed]
16.Ironside JW, Head MW. Variant Creutzfeldt-Jakob disease: risk of transmission by blood and blood products. Haemophilia. 2004;10(Suppl 4):64–69. [PubMed]
17.Castilla J, Saa P, Soto C. Detection of prions in blood. Nat Med. 2005;11:982–985. [PubMed]
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23.Korth C, Stierli B, Streit P, Moser M, Schaller O, et al. Prion (PrPSc)-specific epitope defined by a monoclonal antibody. Nature. 1997;390:74–77. [PubMed]
24.Zanusso G, Liu D, Ferrari S, Hegyi I, Yin X, et al. Prion protein expression in different species: analysis with a panel of new mAbs. Proc Natl Acad Sci USA. 1998;95:8812–8816. [PubMed]
25.Laffont-Proust I, Faucheux BA, Hassig R, Sazdovitch V, Simon S, et al. The N-terminal cleavage of cellular prion protein in the human brain. FEBS Lett. 2005;579:6333–6337. [PubMed]
26.EC. Scientific Veterinary Committee, Report on the risk analysis for colostrium, milk, and milk products (Document N° VI/8197/96 Version J, Final, 1997).
27.EC. Multidisciplinary Scientific Committee, Opinion on the possible risks related to the use of colostrium, milk and products (1997).
28.Statement of the European Food Safety Authority Scientific Expert Working Group on BSE/TSE of the Scientific Panel on Biological Hazards on the health risks of the consumption of milk and milk derived products from goats (2004). Last updated 11. Juli 2006. Available: http://www.efsa.europa.eu/it/science/biohaz/biohaz_documents/709.html. Accessed 2006 Oct 16.
29.Ligios C, Sigurdson CJ, Santucciu C, Carcassola G, Manco GC, et al. PrPSc in mammary glands of sheep affected by scrapie and mastitis. Nat Med. 2005;11:1137–1138. [PubMed]


FIGURES AND TABLES ........SNIP.........END

http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=17183703



TSS
 

Sandhusker

Well-known member
Terry,
From what I have gleaned, detecting the protein doesn't necessarily mean the actual prions are present in the sample (they may be, but we don't know that for sure). Is that correct?
 

flounder

Well-known member
Sandhusker said:
Terry,
From what I have gleaned, detecting the protein doesn't necessarily mean the actual prions are present in the sample (they may be, but we don't know that for sure). Is that correct?



hello sandhusker !

IN SHORT, you are correct, however, here is the the long version :wink:





> Thus we were able to identify prion protein (PrPC)–the precursor of

> prions (PrPSc)–in milk from humans, cows, sheep, and goats.


snip...


> Conclusions/Significance


> In view of a recent study showing evidence of prion replication occurring in the mammary gland of scrapie

> infected sheep suffering from mastitis, the appearance of PrPC in milk implies the possibility that milk of

> TSE-infected animals serves as source for PrPSc.



TO DATE, BSE has not been detectable in milk. NOW, what about BASE and or atypical TSE in bovine?
well, that's another question. and with testing in the pipeline that is much more sensistive, yet to be validated,
low level TSE infectivity in milk is still very much possible. then, what about accumulation ? well, we all can hope
that we die from old age first. PrPSc HAS been detected in mammary glands of sheep affected by scrapie and mastitis. ...tss




studies of interest ;


SEAC 90th meeting held on the 24th Nov $ THE vCJD EPIDEMIC
Thu Dec 1, 2005 10:23
70.110.89.187


1

© SEAC 2005

NINETIETH MEETING OF THE SPONGIFORM

ENCEPHALOPATHY ADVISORY COMMITTEE

The Spongiform Encephalopathy Advisory Committee held its 90th

meeting in Edinburgh on 24th November 2005, and discussed the

following matters:

CURRENT ISSUES

SEAC was informed about the following issues:

• The SEAC Sheep Subgroup will meet on 24th January 2006

to consider emerging scientific developments on atypical

scrapie and possible implications for the National Scrapie

Plan (NSP) and EU TSE roadmap.

• A recent article1 reporting detection of relatively low levels of

abnormal prion in the mammary glands of five Italian farmed

sheep with coincident clinical signs of natural scrapie and

mastitis. Abnormal prion protein was absent in the

mammary gland of sheep free of mastitis or scrapie. SEAC

agreed that the study provided further evidence that

inflammatory diseases can alter the distribution of abnormal

prion protein in infected animals. However, the particular

form of mastitis was rare in the UK and the sheep breed

studied is prone to particular diseases. SEAC considered

the study to be important. However, it would be premature to

come to firm conclusions about the possible implications of

the findings for UK flocks and further investigations should

be undertaken. It was noted that regulations restrict milk

from animals with clinical mastitis from entering the food

chain.

1 Ligios et al. (2005) PrPSc in mammary glands of sheep affected by scrapie

and mastitis. Nature Medicine, 11 published online 04/11/05.

2

© SEAC 2005

snip.......end

http://www.seac.gov.uk/summaries/seac89_summary.pdf


http://www.nature.com/nm/journal/v11/n11/abs/nm1105-1137.html



see full text here ;



PrPSc in mammary glands of sheep affected by scrapie and mastitis.
Ligios C,
Sigurdson CJ,
Santucciu C,
Carcassola G,
Manco G,
Basagni M,
Maestrale C,
Cancedda MG,
Madau L,
Aguzzi A.


http://www.neuroscience.unizh.ch/e/pdf/AguzziV.pdf




2006



"Occurrence and Stability of the BSE Agent in Foodstuff (primarily in Milk and Milk Products) and in the Environment"
For the protection of the consumer a scientifically based risk assessment of animal derived products, particularly milk, meat and products thereof is of prime importance. The knowledge on BSE in this regard is insufficient and also this topic is at present not taken into account in the EU projects on transmissible spongiform encephalopathies. It is also unknown at present, if and to what degree the BSE prions, considered to be very stable, can be degraded or inactivated by microbial processes in the gastrointestinal tract of animals, by fermentation processes during food processing and by the complex microflora of the environment.
The project therefore aims at the improvement of food safety as well as the protection of the consumer and the environment. In order to create the scientific base for a quantitative risk assessment, the following tasks will be solved using modern trace analytical, microbiological and molecular biological methods.
1) Sensitive detection methods for PrPSc in bovine body fluids shall be developed with the main emphasis on the detection of small amounts of the agent in milk but also in blood and meat dripping juice.
2) The microbial degradation of PrPSc shall be studied in order to gain basic knowledge on the stability and fate of the BSE agent in the gastrointestinal tract, in the environment (soil) and in foodstuff (milk and meat products).

Application:
Scientific basis for a rik assessment for food

URL:

http://www.wzw.tum.de/micbio/
Project partners:

Bundesanstalt für Fleischforschung - Institut für Mikrobiologie und Toxikologie
TU München - Institut für Mikrobiologie, Forschungszentrum für Milch und Lebensmittel Weihenstephan
BFAV - Institut für neue und neuartige Tierseuchenerreger
LMU München - Lehrstuhl für Hygiene und Technologie der Milch
Publications:

Biochemical evidence for the proteolytic degradation of infectious prion protein PrPSc in hamster brain homogenates by foodborne bacteria
Müller-Hellwig S, Groschup MH, Pichner R, Gareis M, Märtlbauer E, Scherer S, Loessner MJ
Syst Appl Microbiol 2006 Mar; 29 (2): 165-71

Degradation of scrapie associated prion protein (PrPSc) by the gastrointestinal microbiota of cattle
Christina Scherbel, Rohtraud Pichner, Matin H.Groschup, Simone Mueller.Hellwig, Siegfried Scherer, Richard Dietrich, Erwin Maertlbauer, Manfred Gareis
Vet Res. 37 (2006) 695-703

Cellular Prion Protein in the Bovine Mammary Gland Is Selectively Expressed in
Didier A, Dietrich R, Steffl M, Gareis M, Groschup MH, Muller-Hellwig S,
J Histochem Cytochem. 2006 Jul 24; [Epub ahead of print]

Prion protein expression in bovine podocytes and extraglomerular mesangial
Amselgruber WM, Steffl M, Didier A, Martlbauer E, Pfaff E, Buttner M.
Cell Tissue Res. 2006 Jun;324(3):497-505. Epub 2006 Feb 17

Relevant Branches:

food technology
health

http://www.abayfor.de/forprion/en/projekte_detail.php?pk=657




SPONGIFORM ENCEPHALOPATHY ADVISORY COMMITTEE

Minutes of the open session of the 88th meeting held on 30th June 2005



ITEM 4 – RESEARCH ON ABNORMAL PRIONS IN BOVINE MILK

(SEAC 88/2)



starts at page 6 of 21.......tss



http://www.seac.gov.uk/minutes/final88.pdf



2001




Risk of TSE infection through milk
There never has been any evidence of milk as a method of transfer of scrapie and experiments in which a ewe with scrapie was suckled by the offspring of another sheep showed no indication that any risk was present.
However, it would be surprising if there was no infection present in milk at all simply because of the number of white cells that are present in the milk. At the moment there are no useful inoculation studies published that look for milk infectivity adequately.



--------------------------------------------------------------------------------

No evidence for BSE transmission through milk (17.5.99 as a press release by Consumer Safety and Food)

The Scientific Steering Committee (SSC) sees no evidence for
transmission of BSE through milk. Hence, there is no reason for
restriction on the use of milk, stated the 16 independent scientists in
an opinion on possible vertical transmission of BSE (from mother to
calf) on Friday. The higher infection rate of calves from BSE-infected
cows of between 5 and 15% is rather to be found in other routes of
contagion. The definite mechanisms for vertical transmission still need
to be clarified, they said. With respect to these mechanisms, the SSC
considers that transmission of BSE by artificial insemination is unlikely
for semen derived from BSE-affected bulls early in their incubation
period and that transmission of BSE via embryos is unlikely provided
International Embryo Transfer Society protocols are used.

However, the experts stated that - as a precautionary measure - milk
from BSE-affected cows should be taken out, although this milk is not
proven to be infectuous. The safety of milk is permanently monitored
by the SSC. The Committee also put a preliminary opinion on the
recycling of fallen stock, dead animals (including also ruminants, pigs,
poultry, fish, wild/zoo/exotic animals, laboratory animals, fish, fur
animals and cats) and condemned materials, i.e. animals not fit for
human consumption onto internet for comments of interested parties.
With respect to the high risks of TSEs and unconventional, not yet
identified infectious agents, the present industrial processes of
inactivating conventional infectious agents could not be considered
safe enough. The scientists recommend that animals and SRMs with
TSE or suspected of being so, should be incinerated or burned after
rendering and not be recycled for any direct or indirect use, including
use in cosmetics, pharmaceutical or medical products and devises.
The opinion and the preliminary opinion will shortly be available under
the following address :



However one of the additions to the SSC report was:
"Regarding inoculating cattle i/c with milk, the following comment can
be made: In practice it might prove to be more appropriate / useful to
look (also) at certain fractions of milk and to use transgenic mice
rather than cattle once the model is proven. The aim of the
experiments should be to improve the confidence that milk and
colostrum do not transmit BSE. Colostrum is more important in the
context of protecting animal health and eventually eliminating BSE and
thus a BSE source for humans."



--------------------------------------------------------------------------------

Safety of milk with regard to TSE: State of affairs
(From Food Safety announcement, 2001)
1. Summary

a. Because of the recent evidence of BSE being present in a number
of countries where it had not been detected until recently, Commission
services are frequently invited to express their opinion on the security
status of ruminant milk as a possible source of TSE infectivity.

b. The experimental evidence so far of bovine milk being safe with
regard to BSE risk has been questioned because these experiments
were carried out on mice; it was considered1 that these tests may
have underestimated any possible risk because of the species barrier
from cows to mice. It is noted that milk had the potential to transmit
prion diseases like BSE because it contains a significant component of
leucocytes.

The Commission services therefore invited the Scientific Steering
Committee (SSC) to prepare a state of affairs regarding the available
knowledge and evidence on the safety of milk with regard to TSE risk.

On the basis of the discussion of a report prepared by the TSE/BSE
ad hoc Group, the Scientific Steering Committee (SSC) considers that
the conclusions of the Scientific Veterinary Committee (E.C., 1996),
the Multidisciplinary Scientific Committee (E.C., 1997) and itself (E.C.,
1999), remain valid and that the evidence available to date does not
point at milk or colostrum representing a possible risk. The SSC also
supports the recommendation that for precautionary reasons the milk,
colostrum or milk products from suspect BSE cases should not be
offered for consumption.

No final results of further experiments became recently available and
the SSC confirms the recommendations for research made in each of
the previously listed scientific opinions.

2. Report from the TSE/BSE ad hoc Group

The TSE/BSE ad hoc Group reviewed the information and evidence
available to date. The state-of-affairs report prepared at its meeting of
15 March 2001 on the basis of contributions from various scientists
(see section 3) is presented hereafter.

2.1 The Report of the Scientific Veterinary Committee of 1996
(E.C., 1997)

In its opinion of 1996, the Scientific Veterinary Committee, "having
examined the considerable epidemiological and infectivity data
pertaining to BSE, maternal transmission and milk, concludes that
bovine milk from healthy cows and products derived therefrom which
contain no animal-derived additives can be safely consumed in any
form by any species. There is no evidence that milk transmits BSE
and the Committee considers any risk from milk to be negligible".

"In regard to colostrum, [the Committee] regarded any risks to be
negligible." It further recommended a "Prohibition on the use of milk
(or colostrum) or any product derived from these commodities from
any cow clinically suspected to have BSE for any nutritional or other
purpose. An exception for the suspect's own calf can be made" (...).

2.2. The scientific opinion of the Multidisciplinary Scientific
Committee of 1997 (Will, 1997, E.C., 1997b).

The evidence contained in the full report of the Scientific Veterinary
Committee (E.C., 1996) was reviewed in 1997 by the Multidisciplinary
Scientific Committee (Will, 1997; E.C., 1997) that concurred with the
1996 conclusions.

A maternal cohort study provided statistical evidence of a maternal
risk enhancement (e.g., Donnelly et al, 1997a, b)2, but did not provide
evidence on the risk of transmission by milk as the calves in this study
received only colostrum from their dams and as other explanations
than vertical transmission (for example peri-natal conditions) could not
be excluded.

A serious hint that milk does not transmit the disease was the beef
suckler study (Wilesmith and Ryan, 1997, 1998; Donnelly, 1998)
where no cases were reported although all of them received colostrum
and milk.

2.3. The SSC opinion on vertical transmission (1999)

In its opinion of 18-19 March 1999 on The possible vertical
transmission of Bovine Spongiform Encephalopathy (BSE), the SSC
referred as follows to the work of the UK Spongiform Encephalopathy
Advisory Committee (SEAC):

"As regards the risks from bovine milk, the Scientific Steering
Committee refers to the continuous review by the UK Spongiform
Encephalopathy Advisory Committee (SEAC). SEAC has regularly
discussed the safety of bovine milk in regard to BSE, the last time on
9 November 1998. The latest substantive SEAC view, expressed on
16 April 1997, was that the measures currently in place to protect the
consumer were considered appropriate. (UK law states that milk
derived from BSE affected cattle or cattle suspected to have BSE
shall not be sold, supplied or used for human or animal consumption,
with the exception that it may be fed to the cow's own calf.) SEAC
concluded then (16/4/97) that no evidence had been found to suggest
that milk from any species affected by transmissible spongiform
encephalopathies was infectious. The Committee is keeping the
possible risk infectivity in milk under review and stated most recently
on 14 May 1998 that there was no reason to change their previous
advice on the safety of milk. This advice may need to be updated as
new data and information become available.

However, the Scientific Steering Committee notes that, in the absence
of any infectivity studies on semen, embryos, fetal tissue, milk and
colostrum by i/c inoculation of the homologous species in bovines,
ovines and caprines, and in the absence of all the necessary
experimental and epidemiological data as detailed in the report,
precise estimates of these risks cannot be made."

2.4. Summary of presently available evidence (March 2001):

a. To date, the infectious agent has not been detected by mice
bioassay of milk from humans with kuru or CJD or from cattle with
BSE3 or sheep with scrapie.

b. To date, no mother to child transmissions have occurred in any
animal species used in experimental studies (including primates).

c. One single case has been reported on in 1992 of a 38-year-old
pregnant woman with sporadic CJD whose colostrum was found to be
infected when injected i/c into mice (Tamai et al, 1992). However,
following further morphological examination of fixed mouse brain by
immunohistochemistry for PrPSc , the Japanese authorities concluded
that the published results were invalid as no spongiform change or
PrPSc was found on first passage from human colostrum to mice. The
brain from the second passage (mouse to mouse) did show
spongiform change and PrPSc but this was not attributed to
transmission from the colostrum (Prof. K Yamanouchi, personal
communication to R. Bradley February 1997, in: E.C., 1997)

d. No mother to child CJD transmissions have occurred in kuru or CJD
and neither epidemiological nor experimental studies have
demonstrated the vertical transmission of CJD. (There are a handful of
iatrogenic CJD cases in pregnant women who have delivered children
who remain healthy years afterward - in one case 30 years).

e. To date, no cow to calf disease transmissions has occurred in
association with BSE infected suckler cows.

f. No excess of BSE cases in the offspring of affected animals in the
UK has been observed. Outside the UK, no BSE has been observed
so far in the offspring of BSE cows

2.5. Research on milk.

Experiments to test milk which can use enough volume of milk to be
like the realistic situation are difficult to devise. Currently there seem
not to exist plans to inoculate bovine milk i/c into cattle. However a
PrP study of milk from experimentally infected cattle commissioned by
the UK Food Standards Agency is planned and about to start. A small
scale study is nevertheless presently running at the NPU (N.Hunter,
personal communication, 14.03.01) with scrapie susceptible lambs
removed from their mothers and hand reared No results are yet
available. EC FAIR Project N°CT98-7023 looking among other things
to vertical transmission in scrapie. It is expected that it will also
foreseen to look at the colostrum and milk.

2.6. References:

Bradley, R., 2001. Bovine milk: its safety in regard to risks from the
agents that cause transmissible spongiform encephalopathies (TSE).
Private consultancy report provided on a confidential basis. 9pp.

DONNELLY C.A., FERGUSON N.M., GHANI A.C., WILESMITH
J.W., ANDERSON R.M., 1997b. Analysis of dam-calf pairs of BSE
cases : confirmation of a maternal risk enhancement. Proc. R.
Soc.Lond. B, 264, 1647-1656.

DONNELLY C.A., GHANI A.C., FERGUSON N.M., WILESMITH
J.W., ANDERSON R.M., 1997a. Analysis of the bovine spongiform
encephalopathy maternal cohort study : evidence for direct maternal
transmission. Appl. Statist., 46, 321-344.

DONNELLY, C.A., 1998. Maternal transmission of BSE: interpretation
of the data on the offspring of BSE-affected pedigree suckler cows.
Vet. Rec. 142, 579-580.

E.C. (European Commission), 1997a. Report of 17 Feb 1997. from
the Scientific Veterinary Committee on the risk analysis for colostrum,
milk and milk Products. Document N° VI/8197/96 Version J (Final).

E.C. (European Commission), 1997b. Opinion of 16 May 1997 of
the Multidisciplinary Scientific Committee on the possible risks related
to the use of colostrum, milk and milk Products.

E.C. (European Commission), 1999. Opinion of 18-19 March 1999
of the Scientific Steering Committee on the possible vertical
transmission of Bovine Spongiform Encephalopathy (BSE).

Lacey, R.W., Dealler, S.F., 1994. The transmission of prion disease.
Vertical transfer of prion disease. Human reproduction, 9 (10):
1792-1800. Debate with contributions from P.Brown (pp 1796-1797)
and R.G.Will and J.Wilesmith (pp 1797-1800).

MAFF, 2000. BSE: A Progress Report. June 2000. MAFF, London.

Tamai, Y., Kojima, H., Kitajima, R., Taguchi, F., Ahtani, Y.,
Kawagichi, T., Miura, S., Sato, M., 1992. Demonstration of the
transmissible agent in tissue from a pregnant woman with
Creutzfeldt-Jakob disease. The New England Journal of Medicine, 327
(9): 649.

TAYLOR, D.M., FERGUSON, C.E., BOSTOCK, C.J. and DAWSON,
M., 1995. Absence of disease in mice receiving milk from cows with
bovine spongiform encephalopathy. Vet Rec 136, 592.

WILESMITH J.W., WELLS G.A.H., RYAN J.B.M., GAVIER-WIDEN
D., SIMMONS M., 1997. A cohort study to examine
maternally-associated risk factors for bovine spongiform
encephalopathy. Veterinary Record, , 141, 239-243.

WILESMITH, J.W., RYAN J.B.M., 1998. Comment on Donnelly 1998
(see above)

Wilesmith, J.W., Ryan, J.B.M 1997. Absence of BSE in the offspring
of pedigree suckler cows affected by BSE in Great Britain. Vet.Rec.,
141, 250-251.

Will, R., 1997. Letter of 9 May 1997 to the secretariat of the
Multidisciplinary Scientific Committee.

3. Acknowledgements:

The contributions from the following scientists are gratefully
acknowledged: Dr.R.Bradley, Dr.P.Brown, Dr.D.Heim, Dr.N.Hunter,
Dr.R.Somerville, Prof.Dr.R.Will.

----------------------------------------

1 See also the Prof.M.Ferguson-Smith, Cambridge University,
reported on in the media of 16 January 2001.

2 These and other studies are further reported and commented on in
E.C., 1999.

3 There is no detectable infectivity in bovine milk or mammary gland
collected from clinically-affected, natural cases of BSE following
bioassay using the i/c route in susceptible mice (MAFF Progress
Report 2000). Furthermore, there is no detectable infectivity in milk
collected from clinically affected, natural cases of BSE at early, mid or
late lactation following bioassay by the i/c or by the oral route in
susceptible mice (Taylor et al 1995).



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