Download Veterinary Student Internship Program (VSIP)

Veterinary Student Internship Program (VSIP) Proposal
Project Title: Impact of age in an intra-species challenge of cattle with C-type BSE
Name and title of supervisors:
Dr. Stefanie Czub, DVM/PhD, research manager, UCVM adjunct professor (Production Animal Health)
Dr. Sam Sharpe, DVM, board certified pathologist
Location of Project:
Canadian & OIE reference laboratories for BSE, NCAD-Lethbridge Laboratory
Lethbridge/Alberta
Description of Project:
Summary: This project is designed to answer the question of whether adult cattle are susceptible to BSE
infection by per oral inoculation and if so whether the distribution of infectious prion is similar to cattle
infected as calves. This has implications for disease control measures and regulatory policy.
Background: Bovine spongiform encephalopathy (BSE) is an invariably fatal neurological disease of
cattle and is one of a group of diseases known collectively as Transmissible Spongiform
Encephalopathies (TSEs) along with Scrapie in sheep, Chronic Wasting Disease in wild and farmed
cervids, and Creutzfeld-Jakob Disease in people. TSEs are associated with the accumulation of
proteinase-resistant isoforms of prion protein in the central nervous system.
BSE occurs following the ingestion of a disease associated isoform (PrPsc) of a normal cellular protein
called prion. After ingestion, PrPsc is taken up preferentially into lymphoid tissue associated with the
gut mucosa and primarily replicates here. This explains why ileum and tonsils are included as specified
risk material (see later). Following this, PrPsc enters nerve endings of the enteric nervous system and
makes its way to the brain via the vagal nerve, sympathetic ganglion chain and the spinal cord (Hoffman
et al., 2011). The exact mechanism of prion-associated disease is not known however the currently
accepted ‘Prion-only’ hypothesis asserts that PrPsc induces conformational change in cellular prion
protein, resulting in aggregation of proteinase-resistant PrPsc and cellular dysfunction/death.
BSE was first identified in the UK in 1986 and was linked to the recycling of ruminant tissue in Meat and
Bone Meal (MBM) containing PrPsc from infected cattle. This led to a large scale epidemic resulting in
more than 182,000 cattle confirmed for BSE. Since then BSE cases were subsequently identified in other
countries in Europe, Japan and North America. The first case of BSE in Canada was diagnosed in 1993 in
a cow imported from the UK. The first case of indigenous BSE, in a cow born in Canada was detected in
2003. BSE became a public health concern when exposure to BSE contaminated beef and blood
products was associated with a new variant of Creutzfeld-Jakob Disease (vCJD) which occurred in much
younger people than the established sporadic (sCJD) or familial forms of the disease (Will et al., 1996).
Initial epidemiological studies on the UK BSE epidemic identified the feeding of either tainted milk
replacer or other protein supplements to young calves prior to weaning as the principal source of BSE
infection (Wilesmith et al., 1992) and all BSE challenge experiments utilize young animals (Wells et al.,
2007). These experiments identified the distribution of infectious prion in the tissues of infected
animals. They indicated that some tissues had a much greater propensity to concentrate prion in the
pre-clinical phase of disease. These tissues were then designated as Specific Risk Material (SRM) which
was banned from human and ruminant consumption. This ruminant-to-ruminant ban was implemented
in Canada in 1997 and extended in 2007 to all animal feeds, pet foods, and fertilizers. SRM in Canada
include distal ileum, regardless of age and the skull, brain, trigeminal ganglia, eyes, tonsils, spinal
cord, and dorsal root ganglia in cattle over 30 months of age.
Canada also implemented a surveillance program in 1992 which tested brain material from cattle over
30 months of age displaying clinical signs consistent with BSE. Clinical signs of BSE are related to the
nervous system and include nervous or apprehensive behaviour, gait or posture abnormalities, failure to
rise, teeth grinding, weight loss, or decreased milk production. Clinical signs are not pathognomonic and
can mimic a wide variety of other neurological disease. Veterinarians and producers were therefore
encouraged to submit material from any animal displaying clinical signs that fell under the
`4D`categories i.e. Dead, Down, Diseased, Distressed. If a positive case is identified the birth cohort of
the affected animal which were likely exposed to contaminated feed prior to weaning are tracked down
and destroyed.
It is unknown whether adult cattle are susceptible to per oral infection with BSE and if so whether the
distribution of infectious prion is similar to that in cattle infected as calves.
Project Outline: The focus of this project is on the identification of SRMs in cattle challenged as adults
with BSE. Three cattle were challenged by mouth at 15 months of age with BSE infected material. All
three animals succumbed to progressive neurological disease consistent with BSE. The project will
involve analysing all tissues collected at post-mortem from these animals via immunohistochemistry and
a supersensitive molecular test to detect the presence PrPsc. These results will then be correlated and
compared to previous data to assess whether the definition of SRM is sufficient for animals infected as
adults. The student will gain considerable experience in performing and analysing
immunohistochemistry for PrPsc, performing the Idexx Herdchek BSE antigen ELISA, and collecting and
analysing data.
Experience to be gained in regulatory medicine:
This project is based on the The Government of Canada’s plan to strengthen Canada's world-class food
safety and the CFIA mandate dedicated to safeguarding food, animals and plants, which enhances the
health and well-being of Canada's people, environment and economy.
More specifically, the project will provide exposure to the main focus of the CFIA Science branch by
generating scientific data for BSE risk assessment and structuring BSE trace-out activities; as well as
gaining hands-on experience in laboratory tests; Quality Management; and on-site national and
international teaching/training activities of the BSE Reference Laboratory (BSE-RL), including
participation in technical audits of the BSE program done by foreign countries.
This project aligns a multidisciplinary team with broad expertise in all areas of prion research, including
pathology, biochemistry, molecular biology and in vivo animal models that are handled under BioSafety
Level 3 (BSL-3) conditions. In addition, due to the BSE-RL’s mandate there is a continuous exchange and
transfer of knowledge and technology on a national and international scale; students, scientists, and
technical staff are in the lab for technical training and knowledge expansion regarding regulation of BSE,
BSE surveillance and confirmatory assays; quality management; working in prion certified laboratories,
including performance in high containment laboratory and animal facility.
The respective student will be adapted to the different tasks, balancing seminars and hands-on lab work
to obtain the most effective learning effect and focus on the expansion of hard skills related to the
individual techniques. In addition, the student of this project - with all other summer students – is
required to attend weekly scientific meetings or workshops and to contribute to discussions on similar
or related topics. In addition, the student will have the opportunity to expand individual soft skills within
the local research environment. While integrating into a resident social group, the student will be
influenced by the attitude, responsibility, ethics and social relatedness of the individual members of the
BSE RL’s team. Wherever possible, training will be expanded to the layer of project management that
includes exposure to administration, accounting, quality and lab safety management, as well as dealing
with ethical restraints on animal experiments. The involvement of a BSL-3 laboratory certified for
handling TSE-infected large animals will provide exposure to a one-of-a-kind, state-of-the-art facility. As
a result, student will develop both hard and soft skills necessary to perform innovative research and
help establish well trained individuals in federal career paths in basic and applied research on prions.
The student should take away that the CFIA values scientific rigor and supports professional and
technical competence; and funding of this project is a strategic investment that will inevitably create the
platform for future opportunities to prepare and recruit Canadian veterinary students for a career in
public health or regulatory medicine and ultimately boost the CFIA’s leadership role in science and
CFIA’s vision to excel as a science-based regulator, trusted and respected by Canadians and the
international community.
Deliverables:
Analysis of tissues from experimental animals via molecular testing via Idexx HerdChek BSE antigen
ELISA (including proficiency certification)
Training to proficiency in performing immunohistochemistry (IHC) for detection of PrPsc in tissue section
Training in analysing IHC for detection of PrPsc
Analysis of tissue from experimental animals by IHC for detection of PrPsc
Project Timeline:
Week 1: Orientation and lab safety
Week 2-3: Trimming of SRM tissues for molecular test
Week 4: Perform Idexx HerdChek ELISA
Week 5: Learn to perform immunohistochemistry for the detection of PrPsc in formalin fixed paraffin
embedded tissue
Week 5: Instruction in tissue recognition and slide reading at teaching microscope with pathologists
Week 6-11: Analyse immunohistochemistry slides from experimental animals
Week 12: Data analysis and write-up
Duration and Start of the Project:
12 weeks (preferably end of May to end of August)
References:
Hoffmann, C., Ziegler, U., Buschmann, A. , et al (2007). Prions spread via the autonomic nervous system
from the gut to the central nervous system in cattle incubating bovine spongiform encephalopathy. J
Gen Virol 88, 1048-1055.
Wilesmith, J. W., Ryan, J. B. M. & Hueston, W. D. (1992). Bovine spongiform encephalopathy: case
control studies of calf feeding practices and meat and bone meal inclusion in proprietary concentrates.
Res Vet Sci 52, 325–331.
Wells G. H., Konold T., Arnold M. E., et al (2007). Bovine spongiform encephalopathy: the effect of oral
exposure dose on attack rate and incubation period in cattle. J Gen Virol 88, 1363–1373.
Will RG, Ironside JW, Zeidler M, et al (1996). A new variant of Creutzfeldt-Jakob disease in the UK.
Lancet 347, 921-925.