Download High mortality among young Wetterhoun dogs due to an

High mortality among young Wetterhoun
dogs due to an immunodeficiency
Drs. F. Wempe
9558799
Supervisor: Dr. V.P.M.G. Rutten
Laboratory technician: Ing. P.J.S. van Kooten and Ing. P. Reinink
Table of Contents
Abstract
3
Introduction
Historical survey
About this research project
4
4
5
Immunodeficiency
Primary immunodeficiency diseases
B-cell or humoral deficiency diseases
T-cell or cellular deficiency diseases
Combined deficiency diseases
Phagocytic cell disorders
Complement deficiency diseases
Within this research project
7
7
9
10
11
12
12
13
Materials and Methods
Animals
Hematology
Immune electrophoresis
FACS
14
14
14
14
15
Results
17
17
19
20
24
Hematology
Immune electrophoresis
FACS
Pathological examination
Conclusion
25
Discussion
27
Acknowledgement
28
Appendix
29
References
30
2
Abstract
The Wetterhoun is an old Dutch hunting dogbreed which consists of a very small population.
Inbreeding is a big risk within the population and the Wetterhoun association realizes this
and therefore takes the necessary measures to prevent inbreeding problems. But despite
these measures the Wetterhoun association is since 15 to 20 years dealing with an
unexplained high mortality among the Wetterhoun pups. The young dogs die seven to
twelve weeks of age after a period of diarrhea followed by neurological symptoms. The first
serum immunoglobulin analysis and pathology reports pointed in the direction of a severe
kind of immunodeficiency in these young dogs, but more research was needed.
The aim of this research project was therefore to have a better insight into the origin of this
immunological problem. Blood was collected from litters of Wetterhoun dogs and was
researched for abnormalities that could help find clues about the background of this disease.
The hematology profile of the affected dogs showed a strong decrease in the lymphocyte
numbers. The serum analysis for immunoglobulins IgA, IgM, IgG1 and IgG2 was done and
showed almost no detectable immunoglobulins in the affected dogs. Furthermore the FACS
result showed that almost no T and B cells where present in the blood of the affected pups
and the pathology reports suggested a very severe kind of immunodeficiency in which both T
and B cells where absent. These results strongly indicate that these dogs are dealing with
Severe Combined Imunodeficiency Disease (SCID).
The immunological tests make a SCID diagnosis at an early stage possible but this still leaves
the question of the immunological and molecular origin of the immunodeficiency disease.
To prevent the birth of affected pups in the future, further genetic research is needed. All
different forms of SCID in other dogs and humans, with a matching hematology and
immunoglobulin profile, form good candidates for this genetic research. But because in dogs
only one autosomal form of SCID has been described, this type of SCID is a legitimate first
candidate to start the genetic research. In this form of SCID, which exists in the Jack Russel
terrier, a mutation in the DNA-PKcs gene disrupts the VDJ recombination that is responsible
for the gene rearrangement of the highly polymorphic antigen-recognition regions of the
lymphocytes. This results in a block of the development of the T and B cells in the prolymphocyte stage. But the other proteins operative in the VDJ recombination are also good
candidates. These are Ku70, Ku80, artemis, XRCC4, RAG 1, RAG 2 and DNA Ligase IV.
Furthermore SCID due to a defect in the adenosine deaminase (ADA) also causes comparable
abnormalities in serum and blood. ADA should therefore also be considered as a candidate.
3
Introduction
The Wetterhoun is the oldest dogbreed in The Netherlands. It is a Frisian hunting dog in
former times used especially for hunting ducks near the water. The Wetterhoun dog consists
of a very small population and inbreeding therefore is a big problem for the Wetterhoun and
the Stabij and Wetterhoun association realizes this. Because of this, the breeding is strongly
regulated by several rules to make sure that inbreeding is kept as limited as possible. In spite
of these rules, the association is struggling with problems since the last 15 to 20 years. They
are dealing with very high percentages of unexplained mortality among the puppies at an
age of 7 to 12 weeks. Within an affected litter typically more than one animal dies from
diarrhea followed by neurological symptoms.
Historical survey
In 1992 the Stabij and Wetterhoun association concluded for the first time that there were
problems with high mortality among the young Wetterhoun dogs and started to investigate
these problems. In earlier times the mortality problems were seen as separate problems,
with no common cause. But in 1992 the similarities were seen and the Associating started
researching.
Within an affected litter one or more pups usually develop diarrhea somewhere around
seven weeks of age, shortly after receiving the first vaccination. When treated with antibiotic
this diarrhea disappears and the animal looks healthy again. But a few days later the pup
develops al kinds of neurological symptoms. In most cases the pups can’t keep there heads
straight, and some of the pups spin their body around en others even show epileptic attacks.
At this point the owner often decides to euthanize the animal. In the past years 114 litters
were born of which 25 litters with similar problems, so it is fair to say that at least 10 % of
the litters is affected.
The Stabij and Wetterhoun association has been looking for an explanation for the high
mortality among these young animals. At first veterinarians thought the problems to be
caused by some kind of viral infection, such as the Parvo or Distemper virus, because
symptoms like diarrhea and neurological problems are also found by these viral diseases.
Because the problems typically started shortly after receiving the first vaccination, it was
though that the vaccination was causing or at least initiating the problems. At that point the
Association decided to stop vaccinating the new born. But despite this vaccination-stop for
one year, animals still died from similar symptoms. This proved that it was not just the
vaccination that caused the disease. And for the first time veterinarians and pathologists
started to look for a possible problem in the immune system. Dr. Gerritsen, Veterinary
specialist on internal medicine and Dr. Vos, pathologist at the GD Deventer where the first to
draw this conclusion and they started, supported by the Wetterhoun association, to search
for possible immunological causes.
At this point it was decided to analyse the blood for immunological problems. The blood of
three pups with symptoms was analyzed by immunelectrophoresis. In this test the affected
animals appeared to have no detectable Immunoglobulines; IgM, IgG and IgA. The fact that
the symptoms started to show just after receiving the first vaccination in that sense, may be
caused by the fact that this moment coincides with a critical point in the decrease of the
4
maternal antibodies in the blood of the pups. Somewhere between 6 to 12 weeks the
maternal immunoglobulins decrease to a point that they have no protecting power in the
pups anymore.[12] These results also seem to indicate that there is indeed an immunological
origin at the basis of the mortality problems.
About this research project
During this research project we tried to find out more about the origin of this immunological
problem in the Wetterhoun dogs. Because of the small size of the population, the mortality
problem has a big impact on this breed. Furthermore the timing is very poor. The pups die at
the age of eight to twelve weeks, which is typically the same age at which the pups are
handed over to there new owners. The result being that the new owners receive a perfectly
healthy pup at seven weeks of age, which in the next couple of weeks develops al kinds of
disease symptoms and in the end dies. At that point the owner has paid numerous visits to
the vet without satisfactory results. All of this makes it a traumatizing and frustrating
experience for the new owner and the breeder.
Therefore, for the survival of the Wetterhoun breed and for the owners, breeders and the
pups it is imperative to find the origin of this immune-mediated problem and more
important to find out if it is possible to diagnose the disorder in an early stadium. This way it
might be possible to prevent part of the traumatizing experience for the new owner,
because the pup is kept with the breeder. And this also prevents unnecessary suffering in
the sick Wetterhoun pups and to much emotional binding of the breeder with the affected
pups. Furthermore, knowing which pup is, or which pups are affected can be very useful but
it is even better to look for ways in which it is possible to prevent the disorder, for instance
by breeding adjustments within the Wetterhoun Association.
To find an answer for the different questions above, we looked at similar problems in other
dogbreeds and in other animals and in humans. For instance, in the Jack Russel Terrier, the
Cardigan Welsh Corgi and the Basset Hound the same kind of problems are seen. By looking
at the corresponding characteristics between the immune problems in these types of dogs
and by comparing these to the problems in the Wetterhoun, we tried to find a possible
common source. But because the number of immunodeficiency diseases in dogs is limited
we also had to take into account that there was a possibility that we where dealing with a
new type of immunodeficiency in the dog. Much more research has been done at
immunodeficiency diseases in humans, so we started by looking at these possibilities. More
information about the type of problems in other species can present us with clues about the
source of the mortality problems in the Wetterhoun dog.
Subsequently, to have a clear insight into immunological problem of the Wetterhoun dog,
blood was collected and researched for abnormalities. During the research period six litters
of Wetterhoun pups where born of which 3 pups died from the unknown immunodeficiency
disease. At six weeks of age blood was collected from these young dogs and as many tests as
possible where conducted on there blood. First a hematology profile was made in which
special attention was paid to white blood cell numbers. This was followed by a serum
immunoelectrophoresis to look for abnormalities in the immunoglobulin pattern.
5
Then from a few selected Wetterhoun dogs and some dogs from a different breed, a FACS
(Fluorescence Activated Cell Sorter) was done to collect information about the type of cells
present in the blood of the dogs. The type of abnormalities in the blood of the patients can
provide us with a better insight into the probable cause of the immune-related problems in
the Wetterhoun dogs.
6
Immunodeficiency
As mentioned before, the Wetterhoun dogs that are subjects of this research project, seem
to suffer from some kind of immunodeficiency disease. For a better comprehension of the
basis of this problem, an introduction to the concept of immunodeficiency is essential.
If the immune system in a person is unable to successfully fight infectious diseases, it is
possible that this person is dealing with an immunodeficiency problem. When confronted
with typical signs like repeated infections, which are poorly responsive to therapy, and in
most cases caused by relatively harmless commensal organisms, then there is indeed reason
to believe that the concerning individual is suffering from an immunodeficiency.
Furthermore failure to respond to vaccination and disease resulting from the use of
attenuated live vaccines are characteristics of an immunodeficiency disease.[4, 11]
In humans and animals all kinds of immunodeficiency diseases can occur. They exist in
primary and secondary form. Primary immunodeficiencies or congenital immunodeficiencies
are relatively rare. They can be X-linked or autosomal recessive. The X-linked version is the
most common in humans and therefore most of the people with an immunodeficiency are of
the male gender.[4, 6] Primary immunodeficiencies most often have a genetic basis. These
individuals are born with a defect in there immune system. The specific signs usually develop
at a very young age, as a result of the ongoing disappearance of the maternal antibodies.[12]
Secondary immunodeficiency diseases are acquired later in live. They are caused by for
instance malnutrition, immunosuppressive diseases (AIDS), some kinds of cancer (leukemia)
or the use of particularly medication like chemotherapeutic or immunosuppressive
drugs.[4,6,11]
During this research project disease and premature dead in seven to twelve weeks old
Wetterhoun dogs was investigated. The symptoms of the young dogs were indicative for an
immunodeficiency disease. The dogs showed repeated infections at seven to twelve weeks
of age. This is typically the age at which the maternal antibody level in young dogs reaches a
very low level and the immune system of the young dog is stimulated to produce antibodies
of its own.[12] When investigated, the infections in the pups appeared to be caused mostly
by ordinary organisms of low pathogenicity. In most cases only one or two pups per litter
were affected, not something you would expect when dealing with ‘only’ an infectious
disease. An ‘normal’ infectious disease would cause symptoms in the majority of the pups in
the litter, not in just one or two pups.[4] The symptoms of the dogs and the pathological
reports all pointed in the direction of an inborn error within the immune system, therefore
mainly the primary immunodeficiencies were investigated during this research project.[4]
The primary immunodeficiency diseases
As mentioned previously, congenital or primary immunodeficiency diseases often have some
kind of genetic origin. The hallmark of such an immunodeficiency is a unmistakable increase
of susceptibility to infections at a very young age. The different types of congenital
deficiencies can be grouped by the part of the immune system that is malfunctioning. Most
disorders affect only the functioning of the B-cells and thus cause antibody deficiencies.
Among these B-cell deficiencies, the IgA deficiency is the most common. There are also
7
disorders that impair only the working of the T-cells, the complement system, the natural
killer cells or the phagocytic cells.[4,13]
Classically the primary immunodeficiencies are classified into four or five large groups. The
four groups are; B-lymphocyte, T-lymphocyte, phagocytic cell and complement deficiencies.
But this classification is highly oversimplified because al lot of immunodeficiencies do not fit
in here and other deficiencies overlap between groups. A lot of other classifications are
possible. For instance, it is possible to classify the deficiencies as a function of their
pathofysiology.[15] Furthermore it is also possible to classify the different deficiencies
according to the type and location of the mutation.[10] But to keep matters simple, within
this paper, the originally four large groups were used and one extra large group, the
combined T-and B-lymphocyte deficiencies, was added.
Next, to get a better insight into the probable origin of the immunodeficiency disease in the
Wetterhoun dog, the different primary immunodeficiency diseases in humans and dogs are
described. Together with the various blood tests that where performed during this research
project, the most likely disease and origin may be determined.
[Diagram from: Day. M.J., Clinical immunology of the dog and the cat. P.291]
This diagram shows some possible points of origin of primary immunodeficiencies.
If problems exist at the level of the stem cell (1) then the pluripotent stem cell fails to differentiate.
Abnormalities at a higher level (2) causes failure in the differentiation of the specific stem cells for T and B
lymphocytes. When problems arise at (3) and (4), the development of only the T-lymphocytes (3) or only the Blymphocytes (4) is disturbed. (5) is the point were the B-cells fail to differentiate into plasma cells and at point
(6) the B-cells fail to produce selected class(es) of immunoglobulins. Problems at point (7) causes a failure to
produce functional neutrophils or macrophages and at point (8) failure to produce one or more complement
components. [14]
8
B-cell or humoral deficiency diseases
B-cell deficiencies are the most common of all primary deficiencies in both humans and
animals. They cause antibody deficiencies in a patient. An Individuals with an antibody
deficiency develops problems early in live, shortly after the disappearance of the maternal
antibodies from the blood. In dogs just a very small amount of immunoglobulins is able to
cross the placenta during pregnancy. The pups are therefore born with only 5 to 10% of the
immunoglobulins they need. The rest of the immunoglobulins are ingested with the milk in
the first day’s after birth. These maternal immunoglobulins protect the young dog until six to
fifteen weeks of age, depending on how much immunoglobulins were ingested.[14]
Patients with B-cell deficiencies are especially susceptible to extracellular bacteria, and
therefore suffer from recurrent pyogenic infections. Normally, these types of bacteria are
opsonized by antibodies or complement. This makes it possible for the phagocytic cells to
ingest and eliminate these bacteria. However, when there is a very low level of antibodies
present, the elimination of the extracellular bacteria is seriously impaired and the individual
is much more susceptible to these kind of bacterial infections.[6] In general the resistant’s to
viral infections is not affected in these patients. Antibody deficiencies are in most cases
caused by defects in B-lymphocytes. But because T-cells provide some antibody production
help for the B-cells, it is also possible that a T-cell defect leads to an antibody deficiency.[4]
In humans there are several antibody deficiency diseases described, of which the most
important are X-linked agammaglobulinemia (XLA), Common variable immunodeficiency
(CVID), Hyper IgM syndrome and Selective IgA deficiency. In dogs a selective IgA deficiency, a
selective IgM deficiency, a type of CVID and an IgG deficiency syndrome are
described.[3,4,6,14]
X- Linked agammaglobulinemia (XLA) is caused by a defect in the XLA gene which encodes
for the protein tyrosine kinase, Btk (Bruton’s tyrosine kinase). This defect causes a block in
the B-cell maturation and thus agammaglobulinemia in the patient. The defect is inherited
through the X-chromosome. Healthy female carriers, who have two XX chromosomes of
which one defected, can transmit this defected X-chromosome to there son’s, who have only
one X-chromosome and thus inherit XLA. Therefore only males are affected. Serum levels of
these patients show undetectable IgM and IgA and often very small amounts of IgG. Also are
B-cells strongly decreased or absent in the patients blood. Until this moment there is case of
XLA known in dogs.[3,4,6,14]
In Common variable immunodeficiency disease (CVID) there is a deficiency in IgG and IgA
and sometimes the IgM levels are also low or absent. B cells are in most cases present in
normal amounts , but T cell numbers can be decreased. CVID causes a recurrent bacterial
infections with mainly gastrointestinal symptoms. The genetic basis of CVID is still unknown,
and there is possibility that this is a multigenic disorder. The origin of this disorder lies
probably in the MHC complex and this most likely the same in the Selective IgA deficiency. In
Shar Pei dogs the Selective IgA deficiency has been decribed and there are also cases known
of pups with a late-onset immunodeficiency which strongly resembles CVID in
humans.[3,4,6,14]
9
The Selective IgA deficiency has already been mentioned above. This is the most common
immunoglobulin deficiency but also the most common primary immunodeficiency. The
disease is seen in 1 at 800 people. Somewhere around 50% of the patients show no
symptoms whatsoever. Patients who do have symptoms, show an enhanced susceptibility to
upper respiratory infections. In this deficiency, like in SVID, the genetic basis is also not
known and is probably multigenic and lies in the MHC complex. In patients the serum IgA is
low or absent. When given transfusion, some patients develop anti-IgA in a
transfusionreactions against IgA. Selective IgA deficiency is seen in Irish Wolfhounds, SharPei dogs and some other breeds.[3,4,6,14]
In a Selective IgM deficiency only the serum IgM levels are low while the other
immunoglobulins are present in normal amounts. This sometimes causes severe recurrent
bacterial infections. The deficiency is probably caused by an abnormality in the T-cell help.
Selective IgM deficiency has been reported in Dobermann Pinscher dogs [3,4]
Like in a selective IgA deficiency, the patients of a selective IgG subclass deficiency
sometimes show no symptoms of the disease. And like an IgA deficiency the genetic basis of
the deficiency is probably multigenic. In dogs a IgG deficiency is seen in Weimaraner dogs
and is therefore called ‘ the weimaraner immunodeficiency syndrome’. Dogs experience
more recurrent bacterial infections due to the IgG deficiency.[4,14]
To detect a B-cell defect which causes an immunoglobulin deficiency, a quantification of the
serum immunoglobulins is necessary. By immunoelectrophoresis it is possible to determine
the levels of IgA, IgM, and IgG in the serum of the young dog.[6]
T-cell or cellular deficiency diseases
When an individual is born with a defect in the T-lymphocytes, he or she is especially
vulnerable to viral diseases. The patient will suffer from opportunistic infections which
normally do not cause disease and there is a big risk of auto-immune diseases and
hematological problems.[2] The susceptibility to all kinds of normally harmless pathogens is
the result of poor functioning of the macrophages. The infected macrophages are normally
activated by the activated T-cells who then kill the pathogens. But the defected T-cells can
not activate the macrophages and therefore the opportunistic pathogens can survive and
cause disease. The same kind of T-cell help is critical in the activation of B-cell proliferation.
Therefore a T-cell defect often comes with defects in other cells. And there are not a lot of
deficiencies which are exclusively caused by a sole defect in the T-lymphocytes. Because of
the central role of the T-lymphocytes in the immune response, patients with a deficiency in
this region are susceptible to almost all pathogens.[6]
To detect a defect in the T-cells it is essential to first make a hematology profile. In this
profile, especially the lymphocyte count, will reveal much about a possible T-cell problem.
Furthermore it is possible to do a phenotyping and a calculation of the CD4:CD8 ratio of the
peripheral blood lymphocytes.[14]
10
Combined deficiency diseases
Because the T-cell plays such a central role in the immune respons, a lot of T-lymphocyte
deficiencies result in a combined deficiency of T-cells and B-cells or NK-cells. These
combined deficiencies are often referred to as Severe Combined Immunodeficiency Disease
or SCID. SCID is the most severe type of primary immune deficiency because the patients
shows virtually no immune response whatsoever and are susceptible to almost all
pathogens.[15]
There are again multiple possibilities to classify the different types of SCID. In the first place
it is possible to distinguish two large groups; X-chromosome linked SCID and Autosomal
inherited SCID. Furthermore it is possible to classify SCID according to the molecular origin,
namely the location of the gene defect.[2] And classification according to the defective
mechanism which is causing the disease is also possible. When looking at the mechanisms
that underlie SCID, two main types can be distinguished; (1) SCID due to defective signaling
by γ-dependent cytokines and (2) due to a defective rearrangement of antigen-specific T and
B lymphocyte receptors.[15]
In humans, X-linked SCID due to a mutation in the γ chain of cytokine receptors (IL-2, IL-4, IL7, IL-15) is the most common form. Almost half of all the SCID patients suffer from X-SCID
and these are all male.[7,18] SCID is a recessive inherited disease, which means that an
individual only develops the disease when two defective genes (from both mother and
father) are inherited. In carriers of the disease, who only have one defected gene, is immune
system is working properly and they are healthy. Only when a child has two carriers as
parents, the gene defects cause SCID. But in X-SCID the mutation is localized on the Xchromosome. Females have two X chromosomes but males have only one. Therefore a male
who inherits this defected gene from its mother also inherits X-linked SCID.
In dogs this type of X-linked SCID is described in the Basset Hound and the Cardigan Welsh
Corgi. Patients with X-SCID have strongly decreased T-lymphocyte levels but normal to
elevated numbers of B-Lymphocytes. [4,6,16]
SCID due to an adenosine deaminase (ADA) or a purine nucleotide phosphorylase (PNP)
deficiency are the most common forms of autosomal inherited SCID. SCID is caused by ADA
in 16 % of the SCID cases.[18] In ADA and PNP, enzyme defects cause accumulation of
nucleotide metabolites in T-lymphocytes which are toxic to the developing T-cells. In a white
blood cell count of patients with ADA the T and B-cells are absent and no antibodies are
found. And in patients with PNP the T-cell numbers are decreased but they have a normal
amount of B-cells. The antibody level is decreased. [4,6,16]
A MHC Class II deficiency can also cause SCID because if MHC class II molecules are not
expressed on the T-lymphocytes, CD4 cells cannot be positively selected and therefore they
can not develop. A MHC II deficiency is also referred to as the ‘bare lymphocyte syndrome’,
which is caused by a mutation in one of the genes that are responsible for the regulation of
the expression of the MHC class II genes. A hematology profile will show normal amounts of
circulating T and B- cells. Nevertheless these cells have almost no functional activity.[4,6,16]
Defects in the VDJ recombination of the T and B-cells is also a possible cause of SCID. During
the rearrangement of the immunoglobulin gene segments, the following enzymatic steps are
completed by different genes. A defect in one of these genes is sufficient to completely block
11
the lymphocyte development at the gene rearrangement stage. The genes that are involved
are: RAG 1, RAG 2, Ku70, Ku80, Artemis, DNApk cs, DNA ligase IV, XRCC4. In humans defects
in RAG 1 and 2, Artemis and DNA ligase IV are described. In dogs a mutation in DNApk cs is
described in the Jack Russel. Patients with SCID due to a mutation in one of the genes
involved in the VDJ recombination show lymphocyte counts with almost no T and B-cells.
The serum Immunoglobulins are also strongly decreased or totally absent.[6,16,17]
Phagocytic cell disorders
An immune deficiency can also be caused by a defective phagocytic function. Leukocytes
reach the infection site by emigrating from the bloodvessels to the site of tissue injury. This
complex emigration process involves different molecules for adhesion or migration of the
cells. Deficiencies in any of the steps necessary for the migration of the phagocytic cells can
cause an immunodeficiency. And when the phagocytic cells are not functioning properly a
patient is especially vulnerable to bacterial infections.[4,6]
The leukocyte adhesion deficiency (LAD) is a common phagocytic deficiency among humans.
In LAD the leukocyte function is impaired, this leads to an adhesion and migration inability of
these cells to the sites of infection. The problem is caused by a mutation in the adhesion
molecules. In dogs LAD is named “canine leukocyte adhesion deficiency’ and is seen the Irish
Setter dog.[4,6,16]
In chronic granulomatous disease (CGD) the phagocytic cells ingest pathogens but they are
not able to kill them. In this disorder the phagocytes are not able to produce NADPH
necessary for the antibacterial activity.
Complement deficiency diseases
Defects in complement components significantly increase the susceptibility to infections by
extracellular pathogens and thus cause immunodeficiency disease. This group of circulating
proteins can bind pathogens and form a membrane attack complex. The complement has an
important role as opsonin, which promotes phagocytosis of bacteria. However, the activated
complement system can also cause a lot of tissue damage if it is not regulated properly. As a
result defects in the complement system predispose to infections but also to autoimmune
diseases. Because the complement system can be activated with or without help of
immunoglobulins, it is part of both the innate as well as the adaptive immune system.[4,6]
Defects in C3 or its activation have a big impact, these are associated with serious infections
by extracellular bacteria. The early components (C2, C4) of the complement are responsible
for the elimination of immune complexes and defects in this location can cause autoimmune
diseases. Defects in later components (C5-C9) have less severe consequences, these patients
are exclusively vulnerable to Neisseria.[4,6] In humans, defects can occur in all the proteins
of the complement system. The consequential immunodeficiency depends on the location of
the defect, if the defect exists early or later in the cascade. In Dogs there are reports about a
C3 deficiency which causes susceptibility to bacterial infections.[14,16].
12
Within this research project
The Wetterhoun dogs who are subjects in this research project show a severe kind of
immunodeficiency disease. The affected pups in the litter develop symptoms at very young
age, somewhere between eight to twelve weeks of age. This is typically also the age at which
the maternal antibodies disappear from the blood and the young dog starts producing it’s
own immunoglobulins.[12] The affected pups show severe recurrent infections which cause
diarrhea and neurological symptoms. And not long after the pups die or are euthanized by
the owners because of the persistent severe problems.
When looking at the description of the different Immunodeficiency diseases in humans and
dogs it is most likely that these dogs are suffering from Severe Combined Immunodeficiency
(SCID). This vision is also supported by previous serum immunoelectrophoresis and
pathology reports of affected dogs which where examined in the past. But definite
conclusions can only be drawn on basis of the results of the different blood tests. These are
described next.
13
Material and methods
Immune deficiencies in human and animal often leads to abnormalities in the circulating
blood cells. Therefore a routine analysis of the blood will tell a lot about the probable cause
of the disease. In this case blood was collected from four litters Wetterhoun pups. A full
blood count was done, the blood was tested for antibodies and when possible FACS was
done.
Animals
During the research period blood was collected at six weeks of age, from the six litters of
Wetterhoun pups that where born during the research period. For privacy reasons and for
simplicity, the litters in this report are named A, B, C, D, E and F. Furthermore, the pups in
the litters are numbered : A1, A2 etc. Blood was collected from a total of 32 pups, of which 3
pups in two different litters died from the immunodeficiency. Further more, blood was
collected from the mothers of the litters and when possible from other relatives. For
organizational reasons it was not always possible to run all the tests for all the dogs in this
research project.
Hematology
EDTA Blood was collected from 32 pups and 5 adult Wetterhoun dogs. A full blood count
was done, including an lymphocyte and granulocyte count. The ADVIA TM 120 analyzer was
used for the cell counting using a method based on a two dimensional laser light scatter.
Further more a litter of Cairn Terrier Pups and a litter of Berner Senner Pups from the same
age was also analysed to compare these results with the results of the Wetterhounpups.
Immune electrophoresis
Immune electrophoresis is a combination of two techniques, electrophoresis and immune
diffusion. These techniques where combined by Grabar and Williams in 1953.[5]
There are two separated parts in the immune electrophoresis. In the first part of the
electrophoresis is used to divide the serum, of the patient and of a normal dog, in different
protein fractions. During the second part of the process the antiserum is applied in the agar
and by diffusion the antiserum and the serum of the patient and the normal dog bind and
form bows, the precipitation lines.
The places of the lines are specific for a certain protein. Further more the diffusion distance
and the form of the bow are indicative for amount of protein present in the serum. In this
research project antiserum against specific serums were used; anti-IgA, anti-IgG1, anti-IgG2
and anti-IgM. By comparing the precipitation lines from serum of a normal dog with the
lines from the patients, it was possible to identify a specific protein deficiency with the
immune electrophoresis.
The reagents used for the immune electrophoresis were; 1xPBS, Barbital buffer, 7,9 g
Barbital-Sodium, 5,3 g Sodium-actetate, Broom phenol blue 0,1% solution in 1x Barbital,
Agar 2% in MQ, Agar 1% in Barbital buffer, Normal dog serum, Anti sera dog IgA, IgM, IgG1,
IgG2 and Gelcode Blue Stain.
14
For the electrophoresis part was first a buffer made from the Barbital-sodium, Sodiumacetate and MQ. The pH was brought to 8,2 with HCL. The agar solutions, 1% and 2%, were
made and warmed in the microwave until the solution was clear. The glass slides were
marked and coated with the 2 % agar solution. Then 2,5 ml of the 1 % agar was added to the
slides. After drying punch holes for the sera were made in the agar. In the middle punch
holes 2,5 µl patient serum and 0,5 µl broom phenol blue and in the outer punch holes 2,5 µl
normal serum and 0,5 µl broom phenol blue were applied.
The buffer tanks were filled with 1 x Barbital buffer and a piece of thick paper was used to
make contact between the buffer and the slides. Then electrophoresis was applied for
approximately one and a half hours.
For the diffusion part of the immune electrophoresis grooves were made in the agar coating.
70 µl anti sera was applied by pipette to the grooves and the ant sera was incubated
overnight at room temperature in a humid environment. The next day the glass slides were
washed twice with 1x PBS and then with water. The glass slides were covered with paper
and dried overnight at 65 ⁰C. On the last day the slides were stained with a blue stain for 30
minutes while shaking on the labo-tech RS300. After the staining the slides were washed
with water and dried on the air.
Serum evaluation by immune electrophoresis was done for dogs in the litters A to C, there
mothers and also for a pool of serum from the Cairn Terrier and Berner Senner pups of the
same age as the Wetterhoun pups. The serum was tested for IgA, IgM, IgG1 and IgG2.
FACS
FACS stands for Fluorescence Activated Cell Sorter. The FACSCaliber was used to analyze and
sort the different cells. The cells are labeled with different fluorochromes (different
fluorescing colors) so the fluorescence intensity for the cells can be determined. And then a
suspension of tagged single cells, one by one, is fed to the FACS apparatus. While flowing
through the instrument, in addition to the fluorescence, two types of light scatter are
measured, the “forward Scatter” which measures the diameter of the passing cell and the
“side scatter” which records the cell granularity. The information about the single cells, “the
different events” is displayed by the computer in a scatter dot plot. In this dot plot, each cell
is represented by a dot, positioned on the X and Y scales according to the intensities
detected for that specific cell.[20]
Previously, beforehand the flowcytometry, the patients blood was purified. This purification
is essential because the cells must be in a single cell suspension with minimal aggregation.
First the blood was diluted 1:1 by combining 2 ml tissue medium with 2 ml blood from the
patients. Because EDTA blood was used, heparin was added together with the medium to
prevent blood clotting. Otherwise the calcium in the medium would bind to the EDTA and
this would cause new blood clotting.
After diluting the blood, a sugar gradient (Histopac) is used to divide the different cells from
each other. The 1:1 dilution is necessary because normal blood is too syrupy to use with a
sugar gradient. In the sugar gradient the light lymphocytes will rise until they are above the
sugar gradient and the heavy granulocytes will sink down. The mix is then centrifuged on 800
G for 20 minutes on 20 °C. Centrifuge without a brake to make sure that the cells don’t mix
in the end.
15
Afterwards the interface is removed. The interface contains the cells researched for the
flowcytometry.
For the right adjustment of the FACS, first two adult Wetterhoun dogs and two Cairn Terrier
pups were tested. Then an immunodeficient patient was tested. Further more the blood
from a litter of five healty Wetterhoun pups was tested to compare with the flowcytometry
results from the immunodeficient pup.[21]
The FACS was done according to:
1
PBS
2
CD3
F
3
4
5
CD21 CD3/ CD8
PE
CD21
6
7
CD8/ CD4
CD3 647
8
9
CD3/ IgG1
CD4 PE
10
IgG1
F
11
IgG2
APC
12
CD4/
CD8
Pathology
During the research period, pathology was done for two of the immunodeficient pups that
died during this period.
16
Results
Hematology
EDTA blood was collected from six litters Wetterhoun pups at six weeks of age, litters A to F.
Within our research period, the blood of five of these litters, the litters B, C, D, E and F was
analyzed. For organizational reasons it was not possible to analyze the blood of the pups
from litter A. The ADVIA TM 120 analyzer was used to do a full blood count. At six weeks of
age there were no symptoms of any kind. All the pups were found to be healthy and without
medical problems. But in spite of the healthy looking pups there were already differences to
be seen in the blood cell counting.
The pups in litter B, D, E and F stayed healthy the following period, but one of the pups in
litter C developed the immunodeficiency. After a week this dog developed symptoms of
diarrhea and one week later the dog showed neurological symptoms. At this point it was
decided to euthanize the dog.
In the blood analysis, the results that are higher than a normal adult dog are shown in red
and the results which are lower than the norm are shown in yellow. The healthy pups in
litter B, D, E and F had some results that were higher or lower than the norm. For example
all the pups had lower hematocrit levels and higher lymphocyte levels. This is a normal result
which can be expected from six weeks old dogs.[12]
But in litter C all the pups had a much lower lymphocyte count. In most dogs the levels
stayed above the level of the normal adult dog, but considering the normal level expected
for a six week old dog, this is a very low lymphocyte count for pups this age. And one of the
dogs even showed a lymphocyte count below the norm. It is probable not a coincidence that
this is the dog that was later euthanized.
The increased LUC levels indicate that there were a lot of “Large Unstained Cells”. These are
large cells that can not be characterized further by the hematology apparatus.
Litter B
1
2
3
4
5
6
7
WBC
x10.e9/L
11,2
10,6
17,9
11,2
17,6
13.2
12,5
HCT
L/L
0.315
0.343
0.340
0.317
0.329
0.310
0.289
Lymph
%
34,7
43,1
34,8
50,8
40.0
46,8
34,0
Mono
%
6,4
8,7
7,0
6,9
9,6
6,2
9,8
17
Neut
%
50,4
40,4
50,6
35,4
43,8
39,6
44,9
Eos
%
4,4
4,1
5,0
4,5
4,0
5,7
5,8
Baso
%
2,4
1,5
1,2
1,0
1,2
0,8
1,6
Luc
%
1,7
2,2
1,3
1,4
1,5
0,9
3,9
Litter C
1
2
3
4
5
6
Litter D
1
2
3
4
Litter E
1
2
3
Litter F
1
2
3
4
5
6
7
Normal
WBC
x10.e9/L
6.6
9.3
10.7
9.0
7.1
10.0
HCT
L/L
0.322
0.300
0.302
0.359
0.317
0.282
Lymph
%
24,2
23,7
15,9
31,3
8,8
26,1
Mono
%
9,1
9,7
9.0
9,4
8,9
8,5
Neut
%
60,7
59,9
68,7
51,9
74,6
59,0
Eos
%
1,8
2,3
1,4
2,2
4,2
1,7
Baso
%
1,0
0,8
1,4
2,4
1,1
0,6
Luc
%
3,2
3,7
3,6
2,9
2,3
4,1
WBC
x10.e9/L
10,7
8,5
11,5
10,2
HCT
L/L
0,346
0,357
0,358
0,303
Lymph
%
46,1
42,1
49,5
38,1
Mono
%
9,5
9,3
5,0
5,6
Neut
%
38,8
41,6
39,3
50,3
Eos
%
4,0
5,0
4,4
4,2
Baso
%
0,7
1,2
1,0
0,9
Luc
%
0,8
0,8
0,9
0,8
WBC
x10.e9/L
18,9
13,5
13,3
HCT
L/L
0,366
0,353
0,370
Lymph
%
33,6
42,1
43,6
Mono
%
4,7
4,9
5,5
Neut
%
55,9
49,1
45,3
Eos
%
4,1
2,3
4,1
Baso
%
1,0
0,6
0,7
Luc
%
0,8
0,9
0,8
WBC
x10.e9/L
8,3
13,9
12,3
11,1
12,6
13,8
13,2
HCT
L/L
0,338
0,380
0,363
0,358
0,381
0,355
0,343
Lymph
%
39,0
41,1
43,3
41,9
36,6
37,1
39,7
Mono
%
8,6
4,4
2,5
5,2
4,5
5,6
4,5
Neut
%
48,6
49,0
47,7
47,2
53,4
51,5
49,2
Eos
%
2,3
3,7
3,7
4,0
4,0
4,2
4,9
Baso
%
0,6
0,9
1,1
0,7
0,7
0,6
0,9
Luc
%
0,9
0,9
1,6
1,0
0,8
1,0
0,9
WBC
HCT
x10.e9/L L/L
5,2-13,9 0,3710,570
Lymph
%
11,839,6
Mono
%
3,3-10,3
Neut
%
42,577,3
Eos
%
0-7,0
Baso
%
0-1,3
Luc
%
0-3,0
Hematology litter A
For organizational reasons, it was not possible to do a full blood count from litter A. In stead
100 blood cells were counted from a blood spread.
In consequence, the results of this blood count is a little bit distorted because when cells are
missing, it looks like other cells are more numerous because 100 cells are counted.
But these results are a good back up for hematology results of litter B, C and D.
2
3
4
Lymph
55
1
2
Mono
3
8
15
Staaf
0
8
4
Segment
42
83
78
18
Eos
0
0
1
Baso
0
0
0
Immune electrophoresis
A Serum immunoglobulin assay was done for all the litters. In the healthy litter B, the serum
immunoglobulins were reduced. In most cases IgA was not detectable and IgM and IgG were
reduced. But this appears to be a normal result for six week old dogs.[12] For comparison a
serum immunoglobulin assay was also done for a litter of six week old Cairn Terriers and
Berner Senner dogs from the university clinic. These results show the same reduced levels of
immunoglobulin. But although the levels of some immunoglobulins are reduced, in the
healthy dogs there was always IgM in a detectable level. This proves that the dogs are
capable of making there own immunoglobulin.[12]
In litter A and C, the immunoglobulin levels were even more reduced in all the littermates.
But there almost always was detectable IgM production in the dogs. Except in dogs
numberA3, A4 and C5, this were the dogs that were euthanized because of
immunodeficiency related problems. They had no detectable serum IgA, IgM or IgG1. There
was sometimes some detectable IgG2, but considering the age of the dogs this was probable
of maternal origin and passively derived.
Litter A
IgG(H+L)
IgA
IgM
IgG1
IgG2
1
2
Reduced
Reduced
Reduced
Reduced
Not detectable
Not detectable
Reduced
Reduced
3
4
5
Not detectable
Not detectable
------
Not detectable
Strongly
reduced
Not detectable
Not detectable
Not detectable
Not detectable
Not detectable
Normal
Not detectable
Not detectable
Not detectable
Not detectable
Reduced
Normal
Litter B
IgG(H+L)
IgA
IgM
IgG1
IgG2
1
-------
Not detectable
Increased
reduced
2
--------
Not detectable
Normal
3
-----
Not detectable
Normal
4
-------
Not detectable
5
------
Not detectable
Strongly
reduced
reduced
6
-------
Not detectable
Not detectable
7
-------
Not detectable
Normal
Strongly
reduced
Strongly
reduced
Strongly
reduced
Strongly
reduced
Strongly
reduced
Strongly
reduced
reduced
19
reduced
reduced
reduced
reduced
reduced
reduced
Litter C
IgG(H+L)
IgA
IgM
IgG1
IgG2
1
2
3
4
5
-----------------------------
Not detectable
Not detectable
Not detectable
Not detectable
Not detectable
reduced
reduced
reduced
reduced
Not detectable
Not detectable
Not detectable
Not detectable
Not detectable
Not detectable
6
-------
Not detectable
reduced
Not detectable
reduced
reduced
reduced
Normal
Strongly
reduced
Normal
CT + BS
IgG(H+L)
IgA
IgM
IgG1
IgG2
Cairn
terriers
pool
Berner
Senner
Pool
------
Not detectable
Increased
Not detectable
Reduced
-------
Not detectable
Normal
Not detectable
Reduced
20
FACS
A routine FACS assay was done for the measurement of absolute numbers of white blood
cells, especially the numbers of lymphocytes. This can provide valuable clues about the
cause of the immunodeficiency. For the right adjustment of the FACS other dogs were
tested first. The blood of two full grown healthy Wetterhoun dogs was analyzed by
flowcytometry. 10.000 cells or “events” were counted, but the erythrocytes and the
thrombocytes were kept outside the gate. This way mainly the white blood cells were
counted.[21]
Adult Wetterhoun dog
The adult Wetterhoun dogs showed normal levels of cells. In the lower left corner some
erythrocytes and thrombocytes can still be seen. They were mostly kept out of the gate. The
forward scatter is indicative for the volume of the cell and the side scatter records the
granularity of the cells. The granulocytes show the most granularity and are thus shown as a
group of higher cells. The forward scatter measures the diameter of the cell and places the
bigger cells more to the right, this group of cells are the monocytes. The smaller and
smoother lymphocytes are shown in the marked section in the dot plot.
21
Healthy Wetterhoun pup at six weeks
The FACS dot plot from a pup of six weeks old is different from an adult dog. [12]
The different cell groups are not as recognizable as in the analysis of the adult dog. But all
the different types of cells are without doubt present in the dot plot. Again, the marked
section shows the lymphocytes.
22
Immunodeficient Wetterhoun pup at six weeks
Here we see the FACS dot plot from one of the patients. In the left corner the red blood cells
are shown.
There is also a big cloud of different bigger and more granular cells detectible. In this dot
plot diagram it is difficult to accurately recognize the different cells. But when looking at the
marked section, it is very clear that there are no lymphocytes detected. Because there are
no lymphocytes present, it is no use to differentiate by fluorescence on CD3, CD4, CD8
etcetera.
23
Pathological examination
Macroscopic examination
The pups showed an normal development and muscle conformation, with plenty fat
reserves.
Macroscopically the mandibular and parotidal lymph nodes were of normal size. And also
the thymus was of a normal size. Macroscopically there was not much to be seen.
Histological examination
During the histological examination interesting findings were done in especially the thymus,
the lymph nodes and the spleen. The thymus showed hypoplasia of cortex and medulla and
it lacked the normal corticomedullary demarcation. There were no detectable Hassall’s
corpuscles. Lympoid aplasia was present in Lymphe nodes and in the spleen. The lymph
nodes had a very small cortex and paracortex and lymph follicles were not detectable.
24
Conclusion
At the start of this research project, we set out to find more about the origin of the
immunodeficiency-related mortality among the Wetterhoun pup and to look for ways in
which the immunodeficiency could be diagnosed in a early stage or even, if possible could be
prevented. To find these answers a small literature research was done and furthermore
blood from litters Wetterhoun dogs was collected and analyzed for abnormalities.
During the research period the blood of six litters of six weeks old Wetterhoun pups was
collected and analyzed. As much different tests as possible where done with this blood. For
comparison in de different tests, the blood of a few adult Wetterhoun dogs and that of six
weeks old pups from other breeds was also tested. Among the group of Wetterhoun pups in
the research project, there were three affected pups who died during this period due to the
immunodeficiency disease.
The hematology profile of an affected litter showed decreased lymphocyte numbers in all
the pups compared to what would be expected in six weeks old dogs. And the affected pups
themselves even showed a strongly decreased lymphocyte count.
The serum analysis for immunoglobulins IgA, IgM, IgG1 and IgG2 were significally reduced in
all the Wetterhoun pups, but even more so in the affected pups. The serum analysis of these
dogs showed almost no detectable immunoglobulins. And if immunoglobulins where
detectable in very small levels, this was probable of maternal origin.
The FACS results from the affected dogs showed almost no T or B lymphocytes present. And
the pathology results of the these pups show thymus hypoplasia and lack of normal
corticomedullary demarcation in the thymus. The lymphoid aplasie was also present in the
lymphe nodes and in the spleen. The lymph nodes had a very small cortex and paracortex.
Lymph follicles are not detectable.
The results of the different blood tests and the pathology report all support the vision that
the Wetterhoun dog suffers from a combined T and B-cell deficiency referred to as Severe
Combined Immunodeficiency (SCID). At this moment it is only possible to diagnose this
immunodeficiency in Wetterhoun pups. A positive diagnose can now be made on basis of
the hematology profile with very low lymphocyte count, supported by a serum analysis for
immunoglobulins and FACS results. This diagnosis at an early stage makes it possible to
prevent unnecessary emotional and physical suffering in dog, breeder and owner. But this
solves just part of the problem in the Wetterhoun population. To prevent the birth of
affected pups in the future, further genetic research is needed. Insight in the genetic basis of
this immunodeficiency will present us with possibilities to look for carriers of the disease to
prevent the disease at the breeding level. The different possible types of SCID in humans and
dogs are described previously in the first chapters of this report. These are the most likely
candidates to consider when looking for the molecular origin of this disease.
When looking at these different possibilities it is interesting to see that, although many
different forms of SCID exist in humans, in dogs there are only two different forms known,
namely X-linked SCID in the Basset Hound and the Cardigan Welsh Corgi and autosomal
inherited SCID in the Jack Russel due to a defect in the DNA-PKcs gene.[9] Therefore it is
reasonable to also start by looking at these two forms in the affected Wetterhoun pups. But
as described before, the X-linked SCID only affects male patients. And because in the
Wetterhoun dog the patients are both male and female this form of SCID is not possible. This
25
leaves the form of autosomal SCID due to a defect in the DNA-PKcs gene that exists in the
Jack Russel terrier.[1,8]The DNA-PKcs gene is part of the VDJ recombination pathway. This
pathway is responsible for the gene rearrangement of the highly polymorphic antigenrecognition regions of the lymphocytes and thus accounts for the immunoglobulin diversity
that is necessary for a properly working adaptive immune system. For the production of
these polymorphic antigen-recognition regions the (V)ariable, (D)iversity and the (J)oining
gene segments need to be rearranged by VDJ-recombination.[6,8]
The VDJ recombination starts by recognition and binding of the recombination-activating
genes, RAG proteins 1 and 2, to the recombination signal sequences (RSSs). The RAG
proteins cause a double stranded break in the RSSs and give rise to four loose DNA ends.
This activates the non-homologous end joining (NHEJ) pathway that is responsible for the
DNA repair in the VDJ recombination. In the Jack Russel terrier a mutation in this DNA-PKcs
gene causes a nonfunctional gene resulting in a defective NHEJ pathway and disruption of
the VDJ recombination. A defect in the VDJ recombination has important implications for the
immune system. With a defect in the VDJ recombination, the rearrangement of the antigenrecognition region in T and B cells is disrupted and lymphocyte development is blocked at
the pro-lymphocyte stage.[1,6,8]
A disrupted VDJ recombination causes a very similar kind of SCID as is seen in the
Wetterhoun dogs in this research project. The hematology profile of the Jack Russel terriers
shows very low T and B lymphocyte counts and the serum analyzation for immunoglobulins
shows no detectable IgA, IgM and IgG. This type of SCID and maybe even the location of the
mutation in the Jack Russel terrier are therefore good candidates to start the genetic
research.
26
Discussion
The aim of this part of the research project was to get a better insight in the cause of an
unknown immunodefiency in the Wetterhoun dog which is responsible for a relatively high
mortality rate among Wetterhoun pups. This part, the immunological part of the research
project, ends with the conclusion that the dogs are presumably suffering from a form of SCID
and that genetic research is needed to find both the genetic origin as the precise molecular
location of this disease. But because at this moment only one form of autosomal SCID has
been described in the dog, namely SCID in the Jack Russel terrier due to a defected DNAPKcs gene in the VDJ recombination, this gene is a good candidate to start the genetic
research. Especially because this gene is also an important cause of SCID in other animals
like the Arabian horse and the mouse.[8,9]
Nevertheless, although the DNA-PKcs mutation is a good candidate, there is always the
possibility that the Wetterhoun dog has introduced a new type of SCID in the dog or only a
new mutation. It is therefore important to keep in mind the other possible combined
deficiency diseases and mutations that exist in humans. The research possibilities in humane
medicine are much bigger than those of veterinary medicine and as a result the amount of
mutations that have been researched and described are much larger. When looking at the
different immunological test results in this research project, a few other possibilities have to
be considered for further research.
The DNA-PKcs gene is part of the VDJ recombination in the T and B cells. But there are more
proteins involved in this process and mutations in each of these proteins have the same
result, a disruption of the lymphocyte development at the pro-lymphocyte stage. In mice
and humans mutations in some of these other genes have already been described. Humans
and mice both can have mutations in RAG 1 and 2 proteins.[9,16] And in humans mutations
in DNA-PKcs and Artemis have been seen.[2,16] But the possibility of mutations in one of the
other proteins of the VDJ recombination should also be considered. Besides artemis, RAG1,
RAG2 and DNA-PKcs the other proteins in the VDJ recombination are also possible
candidates. These are: Ku70, Ku80, DNA Ligase IV and XRCC4.[16,17]
Besides other VDJ recombination-mutations, other forms of autosomal SCID should also be
considered as possible candidates. Most types of SCID have hematology profile which differs
from what has been found in the Wetterhoun dogs. These forms of SCID are probably not
very likely in the case of this research project. But there are forms of SCID in which the
hematology results match those of the Wetterhoun dogs in this project. This is the case in
SCID due to a adenosine deaminase deficiency (ADA). In ADA the lymphocytes are
intoxicated due to accumulation of nucleotide metabolites. This causes the same sort SCID
as is seen in the Wetterhoun dogs. Therefore, ADA SCID should also still be considered as a
possible cause of SCID in these dogs. The only difference in ADA SCID is the fact that NK cells
are absent in this disease, while they are present in SCID due to a defect in the VDJ
recombination. To certainly rule out ADA SCID as a candidate the blood has to be analyzed
for the presence of NK cells.
27
Acknowledgement
First I would like to thank Dr. V.P.M.G. Rutten for supervising this project and Ing. P.S.J. van
Kooten and Ing. P. Reinink for assisting the different immunological laboratory activities.
For the genetic part of this research project I would like to thank Dr. P.A.J. Leegwater for
supervising this part and Ing. E.E.C.P. Martens for assisting the different genetical laboratory
activities. Furthermore I like to thank Ing. M. Loohuis, Ing. F.G. van Steenbeek, Mrs A. Slob,
Ing. E.P.M. Timmermans-Sprang and Mrs. J. Wolfswinkel for there help and support in this
research project.
During this research project we where very well supported by the Wetterhoun association
and several veterinarians all over the country. I like to thank the Wetterhoun association for
there support and help during this research, especially Drs. A.M.Lub, Mrs. L. van der Woude,
Drs. J. van der Woude, Drs. Ouwerkerk, Mrs. Wielhouwer, Mr. and Mrs. Vermeulen,
Mrs. Vijverstra, Mr. and Mrs. Hissink, Drs. Schell and Mr. N. Schell and Drs. Van den Berg.
Furthermore I like to thank Dr. E. Teske, Mr. M.W. van Leeuwen and Mrs. B.E. den Hartog of
the Hematology department of Utrecht University, Drs. M.W. de Groot of the pathology
department of the faculty of Veterinary Medicine of Utrecht University, Dr. R.J. Gerritsen
specialist internal medicine and Dr. J. Vos pathologist at the GD Deventer for al the extra
help they provided us with during this research project.
And finally I like to thank my research partner, Drs. B. Verfuurden, for working together,
assisting and supporting me through the project. And I like to specially thank my mother
who was really indispensable in this project and who’s extra efforts made this project
possible, and to my father who’s advise was essential for especially the writing part of this
project.
28
Appendix
The used products for the Fluorescence Activated Cell Sorter (FACS) are:
1
PBS
2
CD3
F
3
4
5
CD21 CD3/ CD8
PE
CD21
6
7
CD8/ CD4
CD3 647
Rat anti dog CD4 647: MCA 1038A647
Mouse anti dog CD3 Fitc: MCA1774F
Mouse anti canine CD21 RPE: MCA178PE
Rat anti dog CD8 RPE: MCA1039PE
29
8
9
CD3/ IgG1
CD4 PE
10
IgG1
F
11
IgG2
APC
12
CD4/
CD8
References
1. Bell T.G., Butler K.L., Sill H.B., Stickle J.E., Ramos-Vara J.A., Dark M.J., Autosomal
recessive severe combined immunodeficiency of Jack Russel Terriers, J. Vet. Diagn.
Invest, 2002. 14: 194-204
2. Bonilla F.A., Geha R.S., Update on primary immunodeficiency diseases, J Allergy Clin
Immunol, 2006. 117; S435-41
3. Tizard I.R., Veterinary immunology: an introduction, Elsevier Publishing: Philadelphia,
2004. p. 413-427
4. Bradley J., e.a., Clinical Immunology, Oxford University Press, New York, 1997. p3-20
5. Grabar R., Williams G., A method permitting the combined study of the
electrophoretic and immunochemical properties of mixture of proteins: Application to
blood serum. Biochem Biophys Ada, 1953. 10: 193—194
6. Janeway C.A., Travers P., Walport M., Shlomchik M., Immunobiology: the immune
system in health and disease, 2001, Garland Science Publishing: New York. p.435-450
7. Buckley R.H., The multiple causes of human SCID, J. Clin. Invest., 2004. 114:1409-1411
8. Meek K., Kienker L., Dallas C., Wang W., Dark M.J., Venta P.J., Huie M.L., Hirschhorn
R., Bell T., SCID in Jack Russell Terriers: A new animal model of DNA-PKcs deficiency,
The journal of immunology, 2001. 167: 2142-2150
9. Perryman L.E., Molecular Pathology of Severe combined immunodeficiency in Mice,
Horses and Dogs, Vet. Pathol, 2004. 41: 95-100
10. Notarangelo L.D., Sorensen R., Is it necessary to identify molecular defects in primary
immunodeficiency disease?, J Allergy Clin Immunol, 2008. 122: 1069-73
11. Reed S.M., Bayly W.M., Sellon D.C., Equine internal medicine, Elsevier Publishing:
St.Louis. p. 37-45
12. Johnston S.D., Root Kustritz M.V., Olson P.N.S., Canine and feline theriogenology,
Saunders Publishing: Philadelphia, 2001. p. 153-158
13. Kumar A., Teuber S.S., Gershwin M.E., Current perspectives on primary
immunodeficiency diseases. Clinical & Developmental Immunology, June–December
2006; 13(2–4): 223–259
14. Day M.J., Clinical immunology of the dog and the cat, Manson Publishing: London,
2008. p. 287-313
15. Fischer A., Primary immunodefieciency diseases: an experimental model for molecular
medicine, The Lancet, 2001. 357: 1863-69
30
16. Notarangelo L., Casanova J.L., Fischer A., Puck J., Rosen F., Seger R., Geha R., Primary
immunodeficiency diseases: An update, J. Allergy Clin. Immunol, 2004. 114: 677-87
17. Janeway C.A., Travers P., Walport M., Shlomchik M., Immunobiology: the immune
system in health and disease, 2001, Garland Science Publishing: New York. p.129-136
18. Simonte S.J., Cunningham-Rundles C., Update on primary immunodeficiency: defects
of lymphocytes, Clin Immunol, 2003. 109: 109-118
19. Ding Q., Bramble L., Yuzbasiyan-Gurkan V., Bell T., Meek K., DNA-PKcs mutations in
dogs and horses: allele frequency and association with neoplasia, 2002. 238: 263-269
20. BD Biosciences, Introduction to flow cytometry: a learning guide, 2000, BD
Biosciences: San Jose. P. 1-44
21. O’Gorman M.R.G., Scholl P.R., Role of flowcytometry in the diagnostic evaluation of
primary immunodeficiency disease, 2002. Clin. Appl. Immunol. 2: 321-335
31