Progress report on the evaluation of the prevalence of anti-neutrophilic cytoplasmic auto-antibodies (pANCA) in the population of Soft Coated Wheaten Terriers in the United Kingdom.
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Report on sampling 5 of the longitudinal study (February 2010)
A familial predisposition for Protein Losing Enteropathy (PLE) and Protein Losing Nephropathy (PLN) has been suggested to occur in Soft Coated Wheaten Terriers (SCWT). The main characteristic
of these two syndromes is the excessive loss of proteins (albumin and globulin) via the gut and the kidneys.
The most important clinical signs are: vomiting, small intestinal diarrhoea, weight
loss, ascites and peripheral oedema. The affected animals are in the age range of 4-6 years and there is a slight predilection of females. As there are no early diagnostic tools for these
diseases available and because PLE/PLN carries a poor prognosis, an early serological marker for predicting these syndromes would be helpful for the prevention of these syndromes in the SCWT.
pANCA has been previously shown to be a useful marker for this disease in SCWT. In a study in 2008, Allenspach et al. have reported that dogs affected with PLE/PLN had a pANCA positive
status and pANCA positive dogs were 3 times more likely to have hypoalbuminaemia.
Moreover, it was noticed that the first pANCA positive result was seen 1-2 years before the onset of the
disease. Thus, detection of pANCA in this breed seems to be a useful test for predicting the later development of PLE/PLN.
In order to estimate the prevalence of pANCA in SCWT in UK, in 2007 our research team carried out a baseline study with SCWT, in which we were able to collect clinical data and blood samples
from 188 healthy dogs. In this study, the prevalence of pANCA in this breed in UK was estimated at 20.7%. Interestingly, this prevalence was much higher than expected.
The samples have been collected during sampling sessions organised by Wheaten Health Initiativethe and the SCWT Club of Great Britain, or directly at the RVC. Since then, a longitudinal study was started
in order to evaluate the usefulness of pANCA as an early serological marker for the development of PLE/PLN. General health status, pANCA status and serum albumin concentrations have been tested
in approximately 80 dogs every six months. For the detection of pANCA indirect immunofluorescence assays were used.
Until now (June 2010), 102 dogs have been enrolled in the longitudinal study. As it was not feasible for all the owners to bring their dogs to all the sampling sessions, we have some missing pANCA results in our longitudinal study.
Thus, 37 out of 102 (36.3%) dogs have been detected as being pANCA negative and 2 (1.96%) pANCA positive at the times they participated in the sampling sessions.
Seven out of 102 (6.8%) have converted from negative to positive,
6/102 (5.9%) from positive to negative and 6/102 (5.9%) have changed their pANCA status twice over the study period.
From the dogs that have participated in all our sampling sessions, 2 (1.9%) have repeatedly tested pANCA positive and 42 (41.2%) have tested negative at all sampling sessions until now.
In the last sampling session 10/63 (15.9%) have been found pANCA positive and 53/63 (84.1%) pANCA negative. (Tables: 1,2).
Table 1 : Number of dogs sampled and tested for the fifth sampling of the pANCA study in February 2010
*no sample available from one dog at this sampling session
Table 2: Frequencies of pANCA positive and pANCA negative blood samples tested in the fifth sampling of the longitudinal pANCA study in February 2010
pANCA positive: 10/63 (15.9%)
pANCA negative: 53/63 (84.1%)
Total number of tested dogs: 63
After the sessions all samples were also tested for their albumin concentration (the reference range for albumin in our laboratory is 28-39 g/l). At the last sampling, 1/62 (1.6%) of the
dogs, had an albumin concentration slightly below the reference range (clinically insignificant), 35/62 (56.5%) were within the range and 26/62 (41.9%) were above it. Dehydration is a reason
for the high results which seem to often see in healthy SCWT. One albumin result from a dog was not available due to insufficient serum sample. (Table 3)
Table 3: Frequencies of Albumin concentrations of the sampling session 5 (reference range: 28-39g/l)
The phenomenon of seroconversion of the pANCA status has also been noticed before in other studies with pANCA using immunofluorescence and it seems possible that it may be due to the level
of the antibody in the blood at each time we test the dog. Thus, in order to determine the true pANCA status of a dog it is important that all study dogs are retested as many times as possible
within the study period.
All results have been communicated confidentially to the owners by mail. In case you have not received your results yet, please do not hesitate to contact the Clinical Investigations Center
(01707 666 605 or 01707 666 223, email@example.com ) at the RVC. If you have any questions regarding the study, or if your dog develops any clinical sign indicative
of possible PLE/PLN please do not hesitate to contact Anna Karagianni* or Karin Allenspach*
After the last sampling session we will be able to shed more light on how useful and accurate pANCA is as an early marker for the detection of PLE/PLN in SCWTs in UK. An important percentage
of SCWTs has already been detected as pANCA positive and regardless of the results of the study, a lot of important information regarding general health and pedigree information on healthy
and affected dogs of this breed will have been collected.
Finally, this study will provide a basis for further investigation in the mode of inheritance for protein losing enteropathy and nephropathy in SCWTs.
As it is of major importance for our study to gather as much information as possibly, we would like to encourage you to participate to our last sampling session in July 2010 even if you
have missed any previous ones.
The study will finish in September 2010 when we will communicate the results to the participant dog owners, the Wheaten Health Initiative and the SCWT Club of GB.
Anna Karagianni and the RVC team, June 2010
Fourth Round Sampling (December 2009) - Tap/Click here (pdf file opens in new window)
Overview of pANCA test results to date (all dogs with ≥ 2 samples)
Progress report on the evaluation of the prevalence of anti-neutrophilic cytoplasmic auto-antibodies (pANCA) in the population of Soft-Coated Wheaten Terriers.
Over the last few years the number of reported cases of protein-losing enteropathies and/or protein-losing nephropathies PLE/PLN in Soft-Coated Wheaten Terriers (SCWT) has
increased in the UK. It has been suggested that a familial link is involved in the predisposition for the disease and given the close links of the UK breed with US breed, concerns were raised
that PLE could also be an emerging problem in UK SCWT population. In the USA it has been estimated that 10%-15% of the SCWT population develop either PLE and/or PLN
at an average age of 4-6 years.
Karin Allenspach and colleagues have shown recently that the presence of perinuclear anti-neutrophilic cytoplasmic auto-antibodies (pANCA) is linked to later onset of PLE/PLN.
This test has been evaluated in a group of 22 SCWT in the USA which were tested for pANCA every 6 months of their lives.
The interesting fact is that a positive pANCA test developed in all dogs on average 2-3 years before showing any signs of illness. This indicates that the
pANCA serum test could be used as an early marker for PLE/PLN in this breed. Positive dogs could then receive appropriate treatment to reduce the risk of developing severe clinical
signs and if a familial pattern is identified, their participation in a breeding programme could be reviewed early in their life, ensuring the continuation of a healthy breed.
In 2007 we carried out a base line study to estimate the number of SCWT in the UK which are pANCA positive. We were able to collect and test blood samples from 189 dogs, and
complete records were available of 188 dogs. The samples were collected either during sampling sessions organised by the SCWT Club GB and the WHI or directly at the Royal
Veterinary College (RVC) in Hatfield. Of the 188 dogs, 39 tested pANCA positive (20.7%).
This prevalence is much higher than what we expected.
If it turns out that the pANCA test is indeed a good predictor for PLE/PLN, then these dogs might be at higher risk to develop protein losing disease later in their life. But by no means
are we certain that a positive test means that the dogs will develop the disease later in life. To be able to assess how much more likely pANCA positive dogs are to develop the
disease compared to pANCA negative dogs, we started a follow-up study in summer 2008.
With this study we hope to re-test 80 pANCA negative and as many positive dogs as possible in intervals of six months over the next 2-3 years.
In June and July of this year, we were able to take blood samples from 82 dogs in two sampling sessions in Weedon Bec, Northampton and St. Leonard's Hall, Oxon. Moreover,
eight samples were taken by private veterinarians and sent to the RVC by post (Table 1), which sums up to a total of 90 samples.
Out of the 90 dogs tested, 77 had been classified as negative and 12 as positive in the initial sample did not have a test result from the baseline study.
Number of dogs sampled and tested for the first round of the longitudinal pANCA study in 2008
All samples collected were tested for pANCA using immunofluorescence, a method that allows labelling of antibodies with fluorescent dyes. Conversion in the pANCA test
result from either negative to positive or from positive to negative was observed in five dogs.
Such fluctuations in antibody concentrations seem to be a physiological phenomenon and have already been observed in the pANCA study in the US . The regular testing over a
prolonged time period will provide conclusive evidence about the definite pANCA status of a dog. This is another reason why it is worthwhile to re-test positive and negative dogs.
In all collected samples we also measured the albumin and total protein concentration. This allows us to detect occult protein-losing disease, as a decreased serum albumin concentration
is a sensitive indicator of possible PLE/PLN.
The reference range for albumin concentration in dogs in our laboratory is 28-39g/l. Among the tested 90 dogs, 33 (36.7%) had normal, 1 (1.1%) had decreased and 55 (61.1%)
had increased albumin levels. Serum albumin is produced in the liver and has important biological functions, such as blood volume regulation or transport of certain substances
(e.g. fatty acids or hormones) in the blood. However, serum albumin concentrations above the reference range occur frequently in healthy dogs and could just be
related to dehydration on the day of serum sampling.
Report on the evaluation of the prevalence of anti-neutrophilic cytoplasmic auto-antibodies (pANCA) in the population of Soft Coated Wheaten Terriers in the United Kingdom.
This study was supported by a grant from the Kennel Club Charitable Trust
Barbara Haesler, Amanda Craig, Barbara Wieland, Carolina Mancho-Alonso, Cathryn Mellersh and Karin Allenspach
Approximately 10% of the population of Soft Coated Wheaten Terriers (SCWT) in the USA and an unreported percentage in the UK develop either protein-losing enteropathies and/or protein-losing nephropathies at an average age of 4-6 years.
The detection of pANCA (perinuclear anti-neutrophilic cytoplasmic antibodies) is a serum test based on evaluation of antibodies which can be easily assessed from about 1ml of whole blood. This test has been
evaluated in a group of 22 SCWT in the USA which were tested for pANCA every 6 months.
The interesting fact is that the positive pANCA result was seen on average 2-3 years before the dogs showed any signs of illness. This means that the pANCA serum test could be
useful in identifying subclinical cases before they become ill. These dogs could then receive early treatment and their participation in a breeding program could be reviewed early in their life.
Our study aimed to estimate how many SCWT in the UK are pANCA positive by obtaining and testing blood samples from a random sample of approximately 200 dogs. Moreover, we
want to find out if pANCA can serve as an early test for the detection of subclinical cases of protein-losing diseases in SCWT and if heritability patterns in the breed can be detected.
In 2007, Dr. Karin Allenspach and her veterinary research team organised 5 blood sampling sessions with the great support of the Soft Coated Wheaten Terrier Club of Great Britain
and the Wheaten Health Initiative. All of these sessions were organized with the help of the Clinical Investigations center at the RVC. In addition, 9 blood samples could be taken
from dogs that were referred to the Queen Mother Hospital or were taken at the local vets and then sent to the Royal Veterinary College in Hawkshead, Hatfield.
In total, 189 dogs could be sampled and tested (Table 1).
Table 1 : Number of dogs sampled and tested for the pANCA study in 2007
In the laboratory, samples were centrifuged within 1 hour and serum was harvested and dispensed into plastic vials. Serum aliquots were properly identified and stored frozen at -20ºC
until testing. The 189 samples were then tested for detection of perinuclear anti-neutrophilic cytoplasmic auto-antibodies (pANCA) using immunofluorescence, a method that allows to
label antibodies with fluorescent dyes. Additionally, serum concentration of albumin was measured to estimate the prevalence of occult protein-losing disease, as a decreased
serum albumin concentration is a sensitive indicator of possible protein-losing disease.
Of the 189 dogs tested:
150 were pANCA negative (79.4%) and 39 were pANCA positive, (20.6%).
Figure 1 shows the age distribution of the tested dogs: Among the negative dogs, 50 were < 4 years and 98 were ≥ 4 years (age of 2 dogs unknown); among the positive dogs,
11 were < 4 years and 27 were ≥ 4 years (age of 1 dog unknown).
Figure 1 : Number of pANCA negative and positive dogs with their age distribution
Serum albumin is produced in the liver and has important biological functions, such as blood volume regulation or transport of certain substances (e.g. fatty acids or hormones) in the blood.
It is important to measure serum albumin concentrations as if this parameter is decreased, then this may be an early indicator of protein losing diseases. However, serum
albumin concentrations above the reference range occur frequently in healthy dogs and could just be related to dehydration on the day of serum sampling.
The reference range for albumin in dogs in our laboratory is 28-39g/l. Among the tested dogs, 146 had normal albumin levels. Among the negative dogs, 5 (3.6%) were found to have
decreased and 21 (14.1%) to have increased albumin levels. Among the positive dogs, 1 2.6%) had decreased and 15 (38.5%) increased albumin levels (Table 2).
Table 2 : Albumin levels of pANCA negative and positive dogs. Reference range: 28-39g/l.
1. Borderline albumin levels, i.e. between 27 and 28g/l.
2. One albumin result was missing
The pANCA results as well as the albumin levels have been communicated to the owners either by mail or email.
In a next step, we would like to follow up about 80 pANCA negative dogs longitudinally, i.e. we plan to test them in 6 months intervals over the next 1-2 years to see if they stay negative.
Additionally, we would like to re-test all positive dogs to confirm the positive results and to monitor the dogs’ disease status over time.
This will help us to asses if there is a link between pANCA results and the later development of protein-losing disease in SCWTs. The longitudinal study will require repeated testing of
the dogs (free of charge) either through attendance at a day testing session or through blood collection by private veterinarians and subsequent postage of the sample to the Royal Veterinary College.
Dr. Karin Allenspach and her research team are currently contacting the owners of both negative and positive SCWT by phone to recruit as many dogs as possible
for the follow-up study. The first follow-up testing session is envisaged to take place in May 2008.
Despite the unknown cause of protein-losing enteropathy and nephropathy, it is believed that there is a familial inheritance in the SCWT. Over the next months, we will examine the pedigrees
of affected dogs to check if there is a familiar pattern of genetic inheritance in the breed.
We would like to thank all participating owners and their dogs for their willingness to help us with our research as well as acknowledge the help of the Clinical Investigations centre at the RVC.
In addition, we would like to thank the Kennel Club Charitable Trust for supporting the study.
We would also like to thank the Soft Coated Wheaten Terrier Club of Great Britain and Wheaten Health Initiative, without their help this study would not have been possible.
We are determined to further investigate protein-losing diseases in the SCWT in order to eliminate these diseases in the future and are convinced that our preliminary investigations represent a first step towards this goal.
Royal Veterinary College, March 2008
Dr. Karin Allenspach, Med. Vet., PhD, DECVIM-CA
Lecturer in Internal Medicine
*If your dog participated in this project and has since died, Dr Allenspach would like to hear from you. Please Contact the Project Coordinator for Dr Allenspach's