PROPOSAL FOR CHRONIC HEPATITIS RESEARCH PROJECT
Principle Researchers:
PENNY WATSON MA VetMB CertVR DSAM DipECVIM MRCVS
And Nick Bexfield BVetMed DSAM DipECVIM-CA MRCVS
The Queen's Veterinary School Hospital
University of Cambridge
Summary of Research:
Chronic hepatitis is a relatively common disease in dogs, but
with the exception of copper storage disease in
Bedlington Terriers, the causes and thus most effective
treatments are poorly understood. The English Springer
Spaniel (ESS) Clubs of both Great Britain and Norway have
noticed a marked increase in chronic hepatitis in the
breed in recent years. Many cases look very similar, both
clinically and histologically and the appearance suggests
a metabolic or infectious cause. The aim of the proposed
research project is first to characterise the disease better
histopathologically and then look for a cause both via
searches for a novel virus (to which ESS may have a breed-
related increased susceptibility) and then via pedigree
analysis and subsequent genetic analysis of affected and non-
affected dogs. The hope is to find the cause of the
condition, which will help treatment of affected dogs, and ultimate-
ly to develop a genetic test to allow it to be removed from
the breed.
Significance of Research:
Chronic hepatitis (CH) is a relatively common disease in dogs
and yet the cause is usually unknown1. Increased
incidences of CH in certain breeds have long been recognised
but, with the exception of a small number of well-
defined copper storage diseases such as those in Bedlington
terriers, there have been no recent advances in the
understanding of these diseases. Treatment therefore remains
non-specific and often not very effective1. The field
of canine genetics has advanced rapidly in recent years2-5.
It is now possible to identify polymorphisms associated
with the genetic basis for a disease with an unknown
aetiology to allow a positional focus on candidate genes for
carriage of the disease associated mutation. This project
represents a very exciting and unique opportunity to inves-
tigate CH in a pedigree breed of dog via microbiology and
genetic analysis with the aim not only of providing a
genetic test to allow it to be eliminated from the breed, but
also to understand the cause and therefore allow more
effective treatment of affected dogs.
Background of Research and Preliminary Work:
Over the last 2 to 4 years, the Health Co-ordinators of the
English Springer Spaniel Clubs (UK)1 noticed an
apparent marked increase in reports of a severe form of
chronic hepatitis in English Springer Spaniels (ESS)
in the UK. It appears to have a high mortality and affects
predominantly young to middle-aged female dogs,
although males are also affected. They were very concerned
and therefore approached Penny Watson at the
Department of Veterinary Medicine, University of Cambridge (DVM
UC) with a view to co-ordinating a project
to find the cause of the condition. The Health Co-ordinators
were particularly keen to undertake genetic studies,
following the recent success of their genetic investigations
of alpha-fucosidosis in the breed, also involving resear-
chers at DVM UC6.
The Health Co-ordinator of the Norwegian Spaniel Club2, a
veterinary surgeon working at the Norwegian Veteri-
nary School, had also noticed a similar disease in ESS in
Norway. Interestingly, ESS in the UK and Norway are very
closely related genetically.
A meeting was held at the KC in London in October 2005 and
again at Crufts in 2006 between the Health Co-ordi-
nators of the English Springer Spaniel Clubs (UK)1 Penny
Watson, The Health Co-ordinator of the Norwegian Spaniel
Club2, Ellen Skancke from the Norwegian School of Veterinary
Science3, Jeff Sampson from The Kennel Club4, and
Nick Bexfield (Crufts meeting only) to discuss the disease
and plan the project. It should therefore be stressed that the
English Springer Spaniel breed clubs, as represented by the
UK Health Co-ordinators1, are very committed to suppor-
ting this project as it was in fact started on their
initiative. The first result of these meetings was a letter to the Veteri-
nary Record7 alerting veterinary surgeons to the disease.
Penny Watson has received many emails and phone calls
about cases since this letter was published.
The instigators of this study are convinced that there is
strong evidence of a distinctive breed-specific hepatitis in ESS,
because of the clinical and histological similarities between
the cases. Subsequent observations of the histology of cases
suggests there may be either a metabolic or a viral cause to
which ESS may have an increased susceptibility. However,
it is very important to ensure that all cases enrolled in the
genetics study have the same phenotype, as far as that is
possible, so collation of clinical and histological details
is an important first part of the study. Even if most ESS with
CH have breed-related disease, there will also be some ESS
with CH which have other causes and these should be
identified and removed from further analysis as much as
possible.
Specific Objectives:
Major funding for a full-scale 3 year study is currently
being sought from various sources. An initial pump-priming
grant of £500 has been awarded by The Alan Emeney Foundation
(TAEF). The specific objectives of this grant are to
allow us to ‘pump-prime’ the study by facilitating collection
and storage of blood and liver samples from as many
affected dogs as possible and collation of pedigree and
history details and histological examination of liver biopsy
samples. This preliminary work is currently being undertaken,
and the value of the pump-priming grant is that it
allows us to continue sample collection before full funding
is obtained for the larger project.
Clinical and histological description will help veterinary
surgeons and owners to identify cases in the future. The
end result of this first year’s project we hope to be a
clinical paper. We have already submitted an abstract to the
European Society of Veterinary Internal Medicine, which has
been accepted for presentation at their annual congress
in September 2007 (abstract attached CLICK HERE).
The aims of the larger project are to build on the samples
and information gained using this pump-primer to search
for a viral cause of CH in ESS and describe the genetic basis
of CH in English Springer spaniels in the UK and Norway.
To achieve this, the clinical and histological details of the
cases must first be analysed as noted above to ensure as far
as possible that all the ESS studied have the phenotype we
are interested in and not another, unrelated form of CH.
Brief description of research methods for pump-primer
project:
Case recruitment: continue to liaise with the breed Health
Co-ordinators in the UK and Norway1,2 and other veteri-
nary surgeons in primary and referral practices to recruit
cases. Veterinary surgeons are indeed contacting us on an
ongoing basis about new cases. We have contacted the major UK
Veterinary pathology laboratories (Finns; North-
western; Abbey; Animal Health Trust, in addition to the UK
Veterinary Schools and referral practices) to alert them
of our project and they have all been very helpful indeed in
sending us blocks of cases. Finns have also put a note on
their histology reports back to vets to alert them about our
research. We will continue to liaise with all of the above
sources in order to obtain further blocks. To be included in
the third year genetics study, affecteds will HAVE to have
had liver biopsies taken, either under ultrasound guidance or
at laparotomy or post mortem. Most cases already have
a biopsy so this is not an onerous requirement.
Description of pathology: all samples will
be stained, examined and reported using a standardised method8 Any cases
with other diseases obvious on histology (e.g. copper or
other storage disease, neoplasia or infections) will be excluded
from further analysis.
Collection of samples for virology: to search for novel
viruses using molecular techniques, we shall need liver tissue
preserved in RNA later rather than formalin from as many
affected and control (unaffected) dogs as possible. We
shall also collect serum samples from the same dogs to search
for anti-bodies to viruses. We shall post out RNA later
to interested veterinary practices to allow them to take
fresh samples when they biopsy the livers of ESS and other dogs.
Pedigree and DNA Analysis:
Collection of DNA for analysis: EDTA blood is currently only
stored from a small number of dogs, but many are
still alive so EDTA blood should be obtained from as many
affected dogs as possible, along with pedigree information.
New cases are now being reported to us at a rate of two or
three per month. We anticipate that this rate will be increa-
sed as more veterinary surgeons in the UK become aware of our
interest. We will also collect samples from Norway
and, if possible, other European countries. There has been no
evidence so far that this disease is a problem in the USA.
Additional samples will be accumulated during the first 12
months of the project, whilst pathological and pedigree
analysis are performed. For the latter, we shall contact
reporting veterinarians and breeders in order to establish health
status for first and second degree relatives of probands. We
shall combine this data with the pedigree information in
order to perform heritability estimates. In addition we shall
use this period to examine mode of inheritance, to see what
is the most appropriate way to analyse our sample collection.
In those cases where EDTA Blood is not obtainable, DNA
will be extracted if possible from formalin fixed sections.
Although this DNA is not suitable for whole genome scans, it
will be used for haplotype and candidate gene analysis once a
confidence interval is established.
Control Samples:
Control blood samples will be obtained from older (> 10 years)
ESS with no clinical evidence of previous or current
liver disease. Full owner consent will be obtained and the
blood samples will be analysed for liver and pancreas en-
zymes. The aim is to obtain between 50 and 100 control
samples.
Expected outcome and applications:
The expected outcome is firstly an accurate and detailed
clinical and histological description of CH in ESS in the
UK and Norway, then identification of any viral cause of the
disease and description of the genetic basis.
These outcomes will allow us both to identify and treat cases
more effectively and also to develop a genetic test for
the breed society to use to help remove this devastating
disease from the breed.
Anticipated problems:
We have the full support of the Breed Clubs
so can foresee few obstacles. The most practical problem will be defining
‘unaffected’ (control) dogs, as we will not be taking liver
biopsies from these animals to prove they have not got chronic
hepatitis. However, since most affected dogs are young to
middle aged, we could define a ‘control’ i.e. normal ESS as
an animal over 10 years of age with normal liver enzymes in
the blood.
Reference List:
1. Watson PJ. Chronic hepatitis in dogs: a review of current
understanding of the aetiology, progression, and treatment.
Vet J 2004;167(3):228-241.
2. Lindblad-Toh K, Wade CM, Mikkelsen TS, et al. Genome
sequence, comparative analysis and haplotype structure
of the domestic dog. Nature 2005;438(7069):803-819.
3. Kirkness EF, Bafna V, Halpern AL, et al. The dog genome:
survey sequencing and comparative analysis. Science
2003;301(5641):1898-1903.
4. Hitte C, Madeoy J, Kirkness EF, et al. Facilitating genome
navigation: survey sequencing and dense radiation-hybrid
gene mapping. Nat Rev Genet 2005;6(8):643-648.
5. Sargan DR, Aguirre-Hernandez J, Galibert F, et al. An
Extended Microsatellite Set for Linkage Mapping in the
Domestic Dog. J Heredity 2006;Submitted for Publication.
6. Skelly BJ, Sargan DR, Herrtage ME, et al. The molecular
defect underlying canine fucosidosis. J Med Genet
1996;33(4):284-288.
7. Watson P, Skancke E, Farstad W, et al. Hepatitis in
English springer spaniels in the UK and Norway. Vet Rec
2006;158(9):311.
8. Rothuizen J, Bunch SE, Charles JA, et al. WSAVA Standards
for clinical and histological diagnosis of canine and
feline liver disease. Oxford: Saunders Elsevier, 2006.
Footnotes:
1 Lesley Bloomfield and Louise Scott Health
Co-ordinators English Springer Spaniel Clubs (UK)
2 Wenche Farstad, Professor, DVM ,PhD Health Co-ordinator,
Norwegian Spaniel Club. Norwegian School of
Veterinary Science, P.O.Box 8146 Dep, N-0033 Oslo, Norway
3 Ellen Skancke, DVM, PhD, Associate Professor Norwegian
School of Veterinary Science, P.O.Box 8146 Dep,
N-0033 Oslo, Norway
4 Jeff Sampson, Kennel Club Canine Genetics Co-ordinator (UK)