Download Replication of scrapie and BSE prions in mites.

DEPARTMENT FOR THE ENVIRONMENT FOOD AND RURAL AFFAIRS
Research and Development
CSG 15
Final Project Report
(Not to be used for LINK projects)
Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
Defra area 6/01
1A Page Street, London SW1P 4PQ
An electronic version should be e-mailed to [email protected]
Project title
Replication of scrapie and BSE prions in mites
SE1829
DEFRA project code
Contractor organisation
and location
Central Science laboratory, Defra
Sand Hutton
York, YO41 1LZ
Total DEFRA project costs
Project start date
£
1/7/1997
Project end date
31/03/01
Executive summary (maximum 2 sides A4)
1.
2.
3.
4.
5.
6.
The only evidence of TSE infectivity in the mouse bioassay was in food deliberately contaminated with
proteins extracted from BSE infected cow brain (1:1, brain protein : mite food). No TSE infectivity was
associated with food contaminated with proteins from scrapie infected sheep brain.
Results from mouse bioassay, Western blot analysis and microscopic examination for scrapie
associated fibrils (SAF) indicated that established mite cultures at CSL were not infected with PrP.
Similarly results for washed mites from scrapie or BSE infected cultures and sub-cultures indicated
that they were not contaminated.
Attempts to detect conserved DNA sequences from mammalian PrP genes in mite DNA extracts were
unsuccessful indicating that mites do not have PrP-like proteins.
The results of this study indicate that even when mites are fed on BSE infected cow brain the mites
(after surface washing) do not carry sufficient amounts of PrPBSE to cause TSE in the mouse bioassay.
In addition, there is no evidence that PrP are replicated in mites of 3 different species that are present
on farms in the UK.
In conclusion, this project has provided data which, when considered in conjunction with the lack of
TSE transmission by mites sampled from UK livestock farms (project SE1828), suggest that mites are
unlikely to be significant vectors or reservoirs of TSE diseases.
CSG 15 (Rev. 12/99)
1
Project
title
Replication of scrapie and BSE prions in mites
DEFRA
project code
SE1829
Scientific report (maximum 20 sides A4)
Background
Transmissible Spongiform Encephalopathies (TSE) are a group of diseases including scrapie in sheep,
Bovine Spongiform Encephalopathy (BSE) in cattle and Creutzfeldt-Jacob Disease (CJD) in humans.
The infectious agents associated with all of these diseases are believed to be Prions ("proteinaceous
infectious particles"), also known as PrP. The proteins are related to endogenous membrane proteins in
mammalian nervous tissue. Although the biochemical and physiological effects of PrP are well
characterised in relation to several pathological conditions, it is not always clear where the prions
originated which cause the domino effect on endogenous proteins. The PrP that cause BSE may have
originated from the brains of scrapie infected sheep or spontaneously mutated cattle, but the source and
causative agent for scrapie is not known. The most likely cause of new variant of CJD was PrP infection
from cattle consumed before MAFF (now Defra) legislation to prevent specified offal from entering the
human food chain.
Evidence in a report in the Lancet (Wisniewski et al, 1996, Lancet, 347, 1114) indicated that when a
mixed sample of mites collected from Icelandic farms, which had a history of scrapie infection in sheep,
were injected into mice some of them developed a spongiform encephalopathy similar to scrapie. The
authors postulated that mites may be involved in transmission of the disease, and that PrP may replicate
in mites. Thus the mite populations in hay fed to sheep may provide a reservoir of infection. The report
also provided evidence that protein preparations from the sample, which included several mite species,
contained protease-resistant proteins that reacted with antibodies raised against known PrP. If mites can
act as a reservoir for the infectious agents, then eradication of BSE could take considerably longer than
anticipated. Mites are contaminants of animal feed and bedding and would be consumed by the animals
as a matter of course. The report from Icelandic farms suggested that mites could form a reservoir for
scrapie over several years. A parallel project (SE1828) has investigated the numbers and species of
mites on 30 sheep farms and 6 dairy cattle farms in the UK and the presence of any infectious material
that could transmit BSE or scrapie.
It is important to know whether or not PrP can replicate in mites. PrP are known to replicate in mammals,
particularly in the lymphoreticular system, and the disease can be passaged through several generations
of laboratory animal using extract of brain and other tissues. There is little known about the lymphatic or
immune systems in mites, and it is difficult to predict how PrP could be replicated. The purpose of this
project was to establish whether PrP can be replicated in mites, and whether they could act as a
reservoir of infectious PrP. This is relevant to Defra policies to eradicate TSEs that may lead to infection
of humans through the food chain.
Methods
Mite cultures
CSL maintains cultures of a number of different mite species, which are pests of stored products. Some
of these have been maintained in the laboratory for over 20 years on a diet made from yeast and
wheatgerm. It is, therefore, unlikely that they have been exposed to PrP from mammalian sources in the
recent past. In the first part of the project proteins were prepared separately from established cultures of
3 different mite species (Acarus farris, Tyrophagus longior and Lepidoglyphus destructor), all of which
were reportedly present in the mixed sample used in the published study from Iceland. Protein fractions
of both mites and feed were examined initially by immunoassay, for the presence of SAF and injected
into mice for assessment of infectivity. This part of the study was completed before known PrP were
brought onto the CSL site to eliminate any possibility of cross-contamination.
Preparation of protein fractions from sheep and cow brain
CSG 15 (1/00)
2
Project
title
Replication of scrapie and BSE prions in mites
DEFRA
project code
SE1829
Frozen brains (25g from control cattle and sheep; 50g from scrapie infected sheep or BSE infected
cattle) were chopped and homogenised in ice-cold acetone. Homogenates were transferred to a flask
and additional ice-cold acetone added to achieve a ratio of 13.3ml of acetone to 1g of brain. The
suspension was maintained on ice for one hour with stirring, filtered and the protein fraction air-dried.
Contamination of mite cultures with PrPSC or PrPBSE
Mites were routinely cultured on wheatgerm flour containing 75% yeast, at 20oC and 90% relative
humidity. A typical mite culture was set up by adding 0.1 g mite adults to food in a 50 ml flask and
incubating for 8 weeks.
Adults (1g) from each species of mites were placed on 5 g food containing brain proteins from BSE
infected cattle, either 1:1 (w/w: high dose) or 9:1 (low dose). Another set of cultures of each species of
mite were set up using food containing the same amounts of protein from scrapie infected sheep brain.
Mites and food were placed in a 50 ml glass conical flask plugged with non-absorbent cotton-wool and
placed in a glass desiccator (6 litre volume) containing 0.5 litre KOH solution of specific gravity 1.1085 to
give 90% humidity. Parallel controls were also set up that contained brain proteins from cattle or sheep
that were not infected with BSE or scrapie.
After 4 weeks of incubation, mites were separated from food as described below. Approximately 0.1g of
mites was added to10 g uncontaminated food for each replicate of each treatment and incubated for
approximately 8/9 weeks. The sub-culturing routine was repeated for a further 2 generations.
Separation of mites from food was carried out by floating on a water surface, followed by filtration
assisted by negative pressure. This was achieved by adding 10 gram of mite culture to a specially
designed floatation apparatus containing about 750 ml of water. The apparatus was attached to a tap
and the level of water was slowly increased. The food particles slowly sank as they were heavier due to
absorption of water, and mites gradually separated from food and floated on the water surface. To
achieve a cleaner separation of mites from food, the water level was raised slowly and mites were
decanted into another similar floatation apparatus through a collection outlet, and subjected to a second
round of separation by floating on water as before. Mites were then decanted into a disposable Hartly
funnel, fitted with a Whatman filter paper number 41, and attached to a one litre Buckner flask for liquidwaste collection. A slight vacuum was applied to Buckner flask to facilitate quick filtration of water whilst
mites, mite-eggs, shed-skins, and a small proportion of food particles were retained on the filter. This
was examined under a microscope to ensure that most food had been removed. An aliquot of mites is
used for further culturing whilst the rest is used in preparation of protein samples (see below).
Homogenates (10% w/v) of mites/food were prepared by grinding in phosphate buffered saline, PBS (pH
7.4) in a pestle and mortar. These crude homogenates were centrifuged at 100g for 10 minutes and the
supernatant stored in sealed ampoules, kept on dry ice, and sent to for mouse bioassy Homogenates for
Western blot analysis were prepared in TBS (10 mM Tris-HCl pH 7.4, 133 mM NaCl) .
Assays for PrP
Preparations of proteins from mites (20l of 10% homogenate) were injected intracranially into 20 mice
as a bioassay for infection leading to development of spongiform encephalopathy. In addition rapid in
vitro immunoassays, based on methodology established for PrP detection in mammalian brain, were
used to establish whether or not laboratory cultured mites contain proteins that are similar to PrP.
Microscopic examination was used to detect “scrapie associated fibrils” (SAF).
Mouse Bioassay of mite samples
Prepared and coded samples were received by VLA from CSL.
Following clearing of bacteriological contamination by heat treatment (80ºC for 15 mins) as necessary,
CSG 15 (1/00)
3
Project
title
Replication of scrapie and BSE prions in mites
DEFRA
project code
SE1829
and where sufficient inoculum was available, the ‘scrapie’ samples were inoculated (0.02ml i.c. and
0.1ml i.p.) into the RIII and VM inbred mouse strains and an F1 cross of the C57bl x VM inbred mouse
strains. Similarly, following bacteriological decontamination, the ‘BSE’ samples were inoculated (0.02ml
i.c. and 0.1ml i.p.) into the RIII inbred mouse strain. Inoculations were carried out according to standard
procedures (Bruce et al 1994). Each inoculum was assayed in 20 mice of each strain.
Mice were monitored on a daily basis during normal husbandry procedures and from 250 days post
inoculation mice were individually clinically examined on a weekly basis and the mice were scored for
signs of neurological disease as follows:
1
=
Normal mouse
2
=
First signs of TSE
Must show major sign marked * OR 2 other signs.
*
+
=
Waddling gait
Rough coat
Dullness about head
Very jumpy
Incontinence
Flattened back
Near to terminal TSE
Must show 2 major signs marked*
*
*
*
*
Marked gait abnormality
Sticky eye discharge and dullness about head
Weight loss
Flattened back
Incontinence
Rough coat
Hunched
This is used to determine the endpoint and is based on established criteria for calculating incubation
period (Dickinson et al 1968). At necropsy the brain of each mouse was removed, fixed in buffered
formalin and processed routinely for histopathological examination. Any mice surviving to the end point
for incubation and expression of disease (650 days for RIII, 900 days for VM and 800 days for F1) were
killed and examined similarly, as were any mice that died or were killed in extremis.
Detection of genes for PrP-like proteins in mites
DNA primers for use in the polymerase chain reaction (PCR) were designed to amplify homologous
sequences of the PrP gene from mammals. PCR amplification was attempted using DNA extracted from
A. farris and T. longior.
Results and Discussion
Mouse bioassay
Infectivity was only detected in the ‘food BSE’ sample (1:1 brain protein : mite food; Table 1). There was
no evidence of infectivity in any of the other samples provided. The previously reported single positive
mouse in the group challenged with ‘G.D. low scrapie’ proved to be negative on more detailed
examination of the brain
CSG 15 (1/00)
4
Project
title
Replication of scrapie and BSE prions in mites
DEFRA
project code
SE1829
Table 1: Summary of results of mouse bioassay of mite samples with confirmed infectivity expressed as
number of positive mice/number surviving into the incubation period range (mean incubation in days ±
SEM)
Inoculum
Inbred mouse strain
RIII
VM
F1 X
Food BSE
2/18 (537.5 ± 113.5)
10/17 (662.1 ± 20.2)
1/5 (803)
Uncontaminated mites
Western blot analysis of protein preparations from uncontaminated mites indicated that proteinase-K
resistant proteins were present in all of the mite cultures, and the mite food, and these were detected by
antibodies to PrP proteins. Although some of the protein bands detected were within the size range (2535kDa) found in TSE infected brain material, the characteristic triple-band pattern was not observed.
Microscopic examination did not detect any evidence for the presence of SAF.
These results indicated that the mite cultures maintained by CSL were not contaminated with PrP
material and are consistent with the absence of infectivity of uncontaminated mites in the mouse
bioassay.
Scrapie and BSE contaminated mite cultures
Western blot analysis of proteins from washed mites from contaminated cultures showed the same band
patterns observed in preparations from uncontaminated mites. Analysis of samples of scrapie and BSE
infected brain tissue did, however, show the presence of PrP protein bands. Microscopic examination did
not detect any evidence for the presence of SAF.
These results indicated that washed mites from deliberately contaminated cultures or sub-cultures were
not infected with either scrapie or BSE PrP and are consistent with the lack of infectivity in the mouse
bioassay.
Detection of genes for PrP-like proteins in mites
Three combinations of primers were used that were designed using conserved sequences from
mammalian PrP proteins. Attempts to amplify PrP like genes from DNA from both A. farris and T. longior
by PCR resulted in amplification of several products. These same products were amplified from the
wheatflour/yeast mite food indicating that the products arose from non-specific amplification of DNA from
the food. PrP like proteins have already been reported in yeasts. It is unlikely that the two species of
mites contained DNA coding for PrP like proteins, or if they do the sequences are different from
mammalian genes.
Please press enter
CSG 15 (1/00)
5