European Society of Veterinary Clinical Pathology
(ESVCP) 8th Annual Congress
Cambridge, UK — September 5–8, 2006
Oral Platform Presentations
1
EVALUATION OF THE SYSMEX XT-2000iV HEMATOLOGY
SYSTEM FOR USE WITH CANINE, FELINE AND EQUINE
HEMATOLOGY TESTING. I. Lilliehook, H. Tvedten. Depart-
ment of Biomedical Sciences and Veterinary Public Health, Swedish
University of Agricultural Sciences, Uppsala, Sweden.
The Sysmex XT-2000iV Hematology System is a modification of the
human XT-2000 and is a ‘‘full count’’ system designed for large and
referral veterinary laboratory use. The instrument has software for
dog, cat, horse and laboratory animals. The methods include
sheath flow DC detection method in RBC and PLT counting and
fluorescent flow cytometery method with semiconductor laser in
reticulocytes, platelets and WBC counting (total WBC and 5 part
differential WBC counting). One may reanalyze samples with
manual gating of various cell populations. This allows selective
counting of cells as one interprets cell populations manually. The
instrument uses 40 ll in capillary mode, 85 ll in manual mode and
150 ll in automated sampler mode. Time for analysis is around one
minute per sample in manual mode. In the current study we
evaluated paired testing of fresh blood samples from diseased
animals with Sysmex XT-2000iV hematology system and Cell Dyn
3500 from 133 dogs, 73 horses and 65 cats. Correlation between the
instruments was between 0.97–1.00 for Hb, RBC, HCT, WBC and
0.90–0.98 for MCV for all three species. For platelets the correla-
tion was 0.99 for dogs and 0.93 for horses. The reticulocyte count
and optical PLT count in cats were not evaluated in this study.
Differential classification of leukocytes was excellent for equine
leukocytes. Correlation was between 0.94–1.00 for neutrophils,
lymphocytes, monocytes and eosinophils. In cats the neutrophil
and lymphocyte population clusters were very near each other,
which often caused inability to count the number of those cells.
About 20% of the cat samples and 13% of dog samples had no
complete differential count reported by the Sysmex. Not reporting
of differential leukocyte counts was especially common with
samples with left shift. The correlation for differential cell counts
for neutrophils was 0.94/0.95 (cats/dogs), for lymphocytes 0.66/
0.39, for monocytes 0.34/0.92 and for eosinophils 1.00/0.96. By
deleting some samples with obvious errors in the separation of the
leukocytes in the graphic results from Sysmex or Cell-Dyn, the
Page 474 Veterinary Clinical Pathology
correlation improved to .0.86. Neither Sysmex or Cell-Dyn
seemed to identify basophils in dogs and cats. In conclusion,
Sysmex XT-2000iV worked very well for hematology samples from
horses, cats and dogs. The main exception was some problems
with the differential count mainly in feline samples.
2
EVALUATION OF EQUINE HEMOGRAMS USING THE
ADVIA 120. A. Giordano1, G. Rossi1, C. Pieralisi2, S.
Paltrinieri1. 1Department of Animal Pathology, Hygiene and
Veterinary Public Health, Unit of General Pathology and Parasitol-
ogy, University of Milan, Italy; and 2Centro Diagnostico Veterinario,
Milan, Italy.
The ADVIA 120 is an automated laser cell counter widely used in
human medicine and in a variety of animal species. Although
specific software for equine haematology is available and in the
literature there are different reports about its use, only a few reports
have been published about the comparison between the ADVIA
120 and other methods for equine hemogram evaluation. In the
present work we compared the performance of the ADVIA 120 to
those of an impedance cell counter (Hemat 8, SEAC) and to the
microscopic evaluation of blood smears. We analysed blood
samples from 114 standardbred healthy horses. Both techniques
are frequently used in equine haematology. The analysis performed
with the ADVIA 120 showed values higher than those of SEAC for
RBC, WBC, Hb, RDW, MCH and MCHC and lower for Hct and
platelets. These differences probably depend on the peculiar
analytic technique of the ADVIA 120 system and, although sig-
nificant, only minimal and poorly biologically important differences
were detected. The greatest differences were in neutrophil and
basophil differentials, which were higher with the ADVIA120 than
microscopic analysis and in lymphocyte percentages which were
lower with the ADVIA 120. The laser measurements should be
considered more reliable because they are obtained on a larger
number of cells. The agreement tests (Passing Bablock and Bland-
Altman plot) showed a linear correlation for RBC and platelet
number, while a constant and proportional bias was present for the
other erythroid and leukocytes parameters. The main advantage
consists in the possible ancillary information about HDW, CH,
CHCM and reticulocyte and related parameter evaluation, usually
not calculated in horses. Repeatability of the ADVIA 120 measure-
ments was not always optimal: the CV related to erythroid
parameters was always less than 3%, while platelets, leukocytes
and reticulocytes CVs were often higher.
Vol. 35 / No. 4 / 2006
 ESVCP Annual Meeting Abstracts
3 reactive hyperplasia, and 3.86 6 1.14 in lymphoma. Mean AgNOR
IMPRECISION OF CANINE MANUAL DIFFERENTIAL LEUKO-
CYTE COUNT: EFFECT OF SMEAR, OBSERVER AND NUMBER
OF COUNTED CELLS. A. Diquélou1, N. Bourgès-Abella2,
C. Picaut1, D. Chafaı̈3,5, A. Geffré1, C. Trumel1, JP. Braun4,5.
1
Internal Medicine, 2Histology, 3Biostatistics, 4Biochemistry, and
5
UMR 181 INRA ENVT, Ecole Nationale Vétérinaire de Toulouse,
Toulouse, France.
Background & objective: The manual differential leukocyte
count (MDLC) is considered the gold standard to which automated
differential counts are compared. However, MDLC imprecision is
estimated statistically, based on the two following assumptions:
leukocyte distribution is homogenous throughout the smear and
one smear is representative of the blood. We investigated if the
leukocyte preparation was homogenous on 3 canine blood smears
and assessed the possible effects of smear, observer and number of
counted cells on MDLC. Methods: Leukocyte preparation: Blood
smears from 3 ‘‘normal’’ dogs were divided into 4 columns and 4
lines delimiting 16 areas (excluding the feathered edge), in which all
cells were counted and differentiated at 3200 or 3400. Differential
count imprecision: All MDLC were blinded. Five routine MDLC (100
cells counted) were performed by 3 observers (2 trained, 1 less
trained) on 10 smears from one ‘‘normal’’ dog and 10 from one dog
with leukocytosis. On the same 20 smears, MDLC was determined
by counting 100, 200, 300, 400 and 500 cells by one observer.
Statistical analysis: Chi-square and ANOVA, using Excel and Systat.
Results: Leukocyte preparation: On the 3 smears, there were
significant differences in MLDC in some areas, columns or lines,
compared to the whole smear, without a common pattern.
Differential count imprecision: Coefficient of variation (CV) for the
neutrophil count was ,7% for the 3 observers, but for eosinophil,
basophil, lymphocyte and monocyte counts CVs were above 48,
100, 19 and 41%, respectively. CVs were slightly lower when
counting 200 or more cells but remained high (27, 86, 13 and 36%,
respectively, with 500 cells counted). Smear effect was statistically
significant for lymphocytes and monocytes in 1 dog, and for
lymphocytes and neutrophils in the other. Observer effect was
significant in 1 dog for the monocyte count. Conclusion: MLDC
is an imprecise method except for neutrophils.
4 a T-cell lymphoma with aberrant expression of CD79a. In the
AGNORS AND KI67 AS MARKERS OF PROLIFERATION IN
CANINE LYMPH NODE CYTOLOGY. N. Bauer, D. Zervos, A.
Moritz. Clinic for Small Animal Clinical Pathophysiology and
Clinical Pathology, Justus-Liebig-University, Giessen, Germany.
The count of Agyrophilic Nucleolar Organizing Regions (AgNORs)
is a useful parameter reflecting proliferation in canine lymph node
cytology and histology (Vajdowich 2004); however, it has not been
compared to other markers of proliferation yet. The scope of this
prospective study was to evaluate the use of Ki67 and AgNORs as
markers of proliferation in canine lymph node fine needle aspirates
(FNAs). Lymph node FNAs of 115 dogs were included in the study.
At least 3 smears were prepared for a May-Grünwald-Giemsa stain,
a Ki67/AgNOR double stain after fixation with Merckofix (Merck,
Germany), and a CD3/CD79b immunostain if cytological exami-
nation was consistent with lymphoma. The dogs were grouped as
normal (n526), reactive hyperplasia (n525), lymphadenitis (n531),
and lymphoma (n533) based on the physical examination of the
lymph nodes and the cytological findings. AgNOR counts/cell and
AgNOR areas/cell and the percentage of Ki67 positive cells were
analysed half-automatically in 100–167 cells using the QWin/
QUIPS software (Leica, Germany). Statistical analysis included an
ANOVA test and an ROC. Mean AgNOR counts/cell were 1.35 6
0.18 in normal dogs, 1.55 6 0.26 in lymphadenitis, 1.65 6 0.31 in
Vol. 35 / No. 4 / 2006 Veterinary Clinical Pathology
area/cell was 40.35 6 4.63 lm 2; 44.73 6 5.29 lm 2, 49.43 6 7.35
lm 2, and 96.62 6 27.88 lm 2, respectively. The percentage of Ki67
positive cells was 2.97 6 0.99% in normal lymph nodes, 5.03 6
3.33% in lymphadenitis, 5.35 6 1.44% in reactive hyperplasia, and
30.23 6 9.08% in lymphoma. All parameters were significantly
higher in dogs with lymphoma than in the other groups (p ,
0.0001). The sensitivity and specificity of the AgNOR count
for diagnosing lymphoma were 91.46% and 96.77% at a cut off
value of ,1.945 AgNORs/cell. The cut off value for the
AgNOR area/cell was ,58.42 lm 2 (sensitivity 96.34%, specificity
93.94%) and ,10.67% for Ki67 (sensitivity 97.56%, specificity
96.97%). The results show that AgNOR and Ki67 counts are good
diagnostic tools for assessment of proliferation in canine lymph
node FNAs.
5
CO-EXPRESSION OF CD3 AND CD79a IN CANINE LYMPHO-
MAS. A. Guija1, W. Gerner2, B. Wolfesberger3, A. Saalmül-
ler2, M. Willmann3, I. Schwendenwein1. 1Central Laboratory,
2
Clinical Immunology, 3Clinic for Internal Medicine and Infectious
Diseases, University of Veterinary Medicine, Vienna, Austria.
The co-expression of CD3 and CD79a in lymphoproliferative dis-
orders is well documented in humans, while in dogs only one re-
cent study demonstrated a high percentage (22%) of lymphomas
with this aberrant expression pattern. We determined the immuno-
phenotype of 24 lymphomas by immunohistochemistry (IHC, 22/
24) and flow cytometry (FCM, 18/24). The expression of the
following canine differentiation antigens was studied with FCM:
CD3, CD4, CD5, CD8, CD21, CD45, CD79a, and sIg. For IHC only
CD3 and CD79a were used. We found 14 B-cell lymphomas (58%),
2 T-cell lymphomas (8%), and 8 lymphomas (30%) with a co-expres-
sion of CD3 and CD79a (mixed phenotype). Co-expression of CD3
and CD79a in FCM was observed in 5/7 cases. In 4 of these the cells
expressed: sIg
, CD21
, CD79aþ, CD3þ, CD4þ, CD5þ, CD8
.
These cases showed a nuclear staining pattern for CD79a in IHC in
contrast to cell membrane staining in positive controls. In two other
cases CD79a was not included in FCM but IHC showed a similar
staining pattern. This pattern as well as the expression of two T-cell
markers combined with only one B-cell marker was suggestive for
remaining case, CD3 and CD79a were expressed in the cell
membranes. The cells expressed (FCM): CD21þ, CD79aþ, CD45þ,
CD4
, CD8
, CD5þ, with a weak CD3 expression. The histological
diagnosis of small non-cleaved cell lymphoma (B-cell origin), the
expression of CD79a in the cell membranes and the expression of
more than one B-cell marker could be an indication for a B-cell
lymphoma with aberrant expression of CD3. Our data indicate that
the presence of lymphomas of mixed phenotype in dogs is more
frequent than previously thought. Since some studies have detected
chromosome aberrations in lymphomas that co-express CD3 and
CD79a and have a poor response to treatment, early recognition of
these lymphomas could be of prognostic value.
6
UTILITY OF FLOW CYTOMETRY IN THE DIAGNOSIS OF
CANINE SMALL CELL LYMPHOMA. F. Riondato1, M.E.
Gelain2, B. Miniscalco1, R. Guglielmino1, S. Comazzi2. 1Dip.
Patologia Animale, Grugliasco, Torino, Italy; and 2Dept. of
Veterinary Pathology, Hygiene and Public Health, Milan, Italy.
Cytologic diagnosis of small cell lymphomas (SC-LSAs) remains
a challenging task. To assess the utility of flow cytometry on fine
Page 475
 ESVCP Annual Meeting Abstracts
needle aspiration biopsies (FNAB) we revisited 21 cases of SC-LSAs
submitted to our laboratories over the last two years. Cases were
classified cytologically, according to the updated Kiel classification,
into: small clear (SCC, 8), lymphoblastic (LB, 4), macronucleolated
medium-sized (MMC, 3), pleomorphic (PSC, 3), lymphocytic (LC, 1
case), prolymphocytic (PRO, 1), and centrocytic (C, 1). Scatter prop-
erties confirmed the cytologic definition of small cell LSAs (FSC
median values significantly different from large cell LSAs). All SCC-,
PSC-, PRO-, and LC-LSAs were of T lineage (13 cases). LB-, MMC-,
and C-LSAs were of B lineage (8 cases), except for one non-B non-T
LB-LSA. All T-LSA were positive for CD3 except for one SCC-LSA
(CD5þ). They were CD4þ, except 3 SCC- (one CD8þ and 2 CD4-
CD8-) and one LC-LSA (CD86). In B-LSA CD21 was always
expressed except for the C-LSA. When used, IgM was always posi-
tive, while IgG was mostly negative. Frequent aberrant expres-
sion of antigens in T-LSAs (9/13), mostly in SCC-LSAs (6/8), and
frequent CD34 expression in B-LSAs (3/4) were evident. In par-
ticular, the expression of panleukocyte markers (CD45, CD18) was
frequently diminished or absent (7 cases, all T-LSA but one). The
results show that small cell LSAs are mainly T-cell tumours often
presenting alterations in antigen expression. We suggest that the
detection of aberrant qualitative and/or quantitative expres-
sion of lymphoid markers could be indicative of clonal expansion.
Therefore, flow cytometric analysis could be a powerful tool to
confirm a tentative cytologic diagnosis of lymphoma since FNAB is
minimally invasive in comparison with other diagnostic procedures
commonly used to confirm small cell LSAs (surgical biopsy).
7 May-Grünwald-Giemsa, although some were received pre-stained
CONCENTRATION OF C-REACTIVE PROTEIN IN DOGS WITH
TREATED AND UNTREATED LYMPHOMA: AN INDICATOR OF
REMISSION STATUS? L. Nielsen, P.D. Eckersall, D. Mellor, L.
Bence, J.S. Morris. Faculty of Veterinary Medicine, University of
Glasgow, Glasgow, UK.
The acute phase protein C-reactive protein (CRP) has shown
potential as a diagnostic and prognostic marker in human patients
with various neoplasias including Non Hodgkin’s lymphoma. We
measured CRP in dogs with treated and untreated lymphoma,
to assess whether chemotherapy would alter the concentration of
CRP and whether the initial CRP affected survival. Diagnosis was
obtained by biopsy and patients were staged and treated with
a standard multidrug chemotherapy protocol. Serum samples were
obtained from all dogs at the point of diagnosis and before each
treatment. CRP was measured by ELISA (reference range for normal
dogs ,10 mg/l). Clinical response to treatment was assessed as
complete remission, partial remission, stable disease or progressive
disease. Statistical analysis was performed using MinitabÒ 14.
Twenty-one dogs with untreated lymphoma were included result-
ing in 227 serum samples. At first presentation, only 21% of dogs
had CRP within the normal range compared to a significantly
higher proportion of dogs (84%), when in complete remission (p ,
0.001). There was a significant difference in the median CRP before
treatment and when complete remission was achieved (p , 0.01). A
significant difference was also obtained between dogs in remission
(complete or partial) and those with progressive disease (p , 0.001).
However, there was no difference in CRP between dogs in clinical
remission and those in partial remission (p50.34). There was no
difference in the change of CRP after administration of vincristine,
cyclophosphamide or anti-tumour-antibiotics. For patients with a
high (.24 mg/l, n510) versus low (,24 mg/l, n511) pretreat-
ment CRP the median survival time was 229 days and 286 days,
respectively. This was not significant. In conclusion, CRP may be
useful to see whether the patient is in remission or relapsing under
treatment. Different chemotherapy agents do not have different
Page 476 Veterinary Clinical Pathology
effects on CRP and the pre-treatment CRP does not predict patient
survival time.
8
FELINE MYELOMA RELATED DISORDERS: CYTOLOGICAL
ANALYSIS OF 13 CASES. P.J. Mellor1, R.M. Powell1, J. Archer1,
E.J. Villiers1, S. Murphy2, G. Polton3, C. Belford4, D.J.
Argyle5, M.E. Herrtage1, M.J. Day6. 1Department of Veterinary
Medicine, University of Cambridge, Cambridge; 2Animal Health
Trust, Kentford; 3Davies Veterinary Specialists, Hitchin; 4Cytopath,
Ledbury; 5Royal (Dick) School of Veterinary Studies, University of
Edinburgh; 6Department of Clinical Veterinary Sciences, University
of Bristol, UK.
The myeloma-related disorders (MRD) derive from the neoplastic
transformation of a plasma cell or its immunoglobulin secreting B-
lymphocyte precursors are rare in the cat. The aims of the study
were to describe the cytological features of a large series of cats with
MRD, to devise a simple and reproducible morphological class-
ification system and to assess cell morphology with respect to tum-
our location. Cases were sourced from all major public and private
UK referral centres and laboratories. The inclusion criteria were the
identification of plasma cell, plasma cell-like or alternatively
lymphoid morphology so long as the latter was accompanied by
hyperglobulinaemia (total serum globulin .52 g/L) attributable to
a paraproteinaemia, as evidenced by serum protein electrophoresis.
The morphological diagnosis was confirmed by a joint-panel of
cytologists (JA, EJV, RMP, PJM) following review of the original
cytological specimens. Preparations were optimally stained with
with Romanovsky stains. Reports for 17 cases were obtained over
the period 1990–2004, of which 13 cases had original specimens that
were confirmed at review. The median age of affected cats was 12
years (range 4–14.75 years), 77% were domestic short haired cats,
38% male, 62% female. Twelve of 13 cases underwent serum
biochemical exams revealing hyperglobulinaemia in all cases and
serum protein electrophoresis demonstrated a paraproteinaemia in
all 12 cases. Plasma cell neoplasia of an abdominal organ accounted
for 46% of all MRD cases at initial presentation with half of these
cases having cytological evidence of multi-organ involvement. The
liver was the most commonly affected sampled abdominal organ
(n55), followed by the spleen (n53) and intestine (n51). All six
cases with abdominal organ involvement had bone marrow
aspiration carried out. Four cases had no evidence of concurrent
marrow involvement (,20% plasma cells) and were classified as
non-cutaneous extramedullary plasmacytoma cases (NCEMP) and
n52 had concurrent marrow infiltration. An additional 5 cases had
marrow infiltration, and a further 2 cases had cutaneous masses—
but these groups did not undergo concurrent cytological examina-
tion of other organs. Tumours were classified as follows: mature
plasma cell type (n59), proplasmacytic (n53), plasmablastic (n51),
mixed cell type (n50) and lymphoid (n50). There was no as-
sociation between tumour location and plasma cell type. In ani-
mals with multi-organ involvement, there were no differences in
morphologic categorization of the MRD between different tissues of
the same animal when evaluated by cytology alone. Giant myeloma
cells (not previously reported in the cat) with large, segmented
nuclei, were observed in one case. In man, mature plasma cell type
tumours are more frequently associated with intra-medullary
disease whereas altered morphology to, e.g., plasmablastic type is
associated with extra-medullary metastases and a worsened prog-
nosis. By contrast, in this study, we report that there was no
correlation between anatomical location and plasma cell morphol-
ogy. Specifically, an extramedullary location did not correlate with
more aggressive cellular morphological features in the cat. This is
further evidence for the hypothesis that a primary extramedullary
Vol. 35 / No. 4 / 2006
 ESVCP Annual Meeting Abstracts
origin for the neoplastic transformation of plasma cells is more
likely in the feline species.
9 in CRP, Hp and fibrinogen and a significant decrease in albumin
CAN CANINE CEREBROSPINAL FLUID PROTEINS BE MEA-
SURED BY THE VETTEST UPRO SLIDES? A. Geffré1, C.
Trumel1, J.P. Braun2. 1Département des Sciences Cliniques and
2
UMR 181 Physiopathologie et Toxicologie Expérimentales ENV-
INRA, Ecole Nationale Vétérinaire, Toulouse, France.
Background: Evaluation of Cerebrospinal fluid (CSF) is a basic
examination in neurology. In-clinic evaluation is based on urine
test strips, which have controversial diagnostic value around the
decision limit (0.3 g/L). The VetTest UPRO slides (IDEXX) are
designed to measure urine protein concentration in the same range
as can be expected in CSF. Objective: The purpose of this study
was to compare results obtained with a validated laboratory
technique with results of the VetTest UPRO slides. Methods:
Duplicate analyses of 149 frozen canine CSF were performed by
a manual pyrogallol red (PR) technique (Secoman S500, Seli &
Protéines Urinaires, Sobioda) and by the VetTest. Accuracy of PR
was controlled before each run with a control urine (Uritrol, 1 g/L,
Sobioda); between-series imprecision was 4.4% (0.3 g/L). Results
were compared by standard statistical procedures with a set of
macroinstructions for Excel (Analyse-It). Results: The Spearman
correlation was good (r50.84). Passing-Bablock agreement equation
(95% CI) was TPVT50.82 (0.74/0.89)  TPPR – 0.02 (
0.04/0.01).
Total imprecision (n533; 1.0 g/L) was high (CV527%). VetTest
results were significantly lower (paired t-test, P , 0.0001). Bias was
proportional, ranging from 0.0 (0.1/0.2 g/L) to 0.6 g/L (1.0/4.5 g/
L) (Mann-Whitney test, P , 0.05). At the 0.3 g/L threshold, 19
results were not similarly classified by the two techniques; after
correction for bias and taking imprecision into account, only 5
VetTest results (4%) were classified as ‘‘normal’’ when CSF proteins
were high by PR. Discussion & Conclusion: Canine CSF proteins
can be easily and rapidly measured with the VetTest UPRO slides.
However, imprecision and accuracy of the slides are responsible for
a limited number of clinical misclassifications around the decision
limit. Cautious use of results in this zone should be made.
10
ACUTE PHASE PROTEINS IN DOGS WITH HYPERADRENO-
CORTICISM: A PARTICULAR PROFILE? M. Caldin1, S. Tasca2,
E. Carli2, C. Patron2, S. Martinez-Subiela3, J.J. Ceron3.
1
Clinica Veterinaria Privata San Marco, Padova, Italy; 2Laboratorio
dAnalisi Veterinarie ‘‘San Marco’’, Padova, Italy; and 3Animal
Medicine and Surgery Department, Faculty of Veterinary Medicine,
Murcia, Spain.
Introduction: Despite their great sensitivity as biochemical
markers of inflammation acute phase proteins (APPs) are still not
extensively applied in canine routine practice. One of the causes
for this situation, in addition to the few and relatively expensive
commercial kits available for its analysis, can be the insufficient
numbers of studies about the specific behaviour of the different
APPs in individual diseases. In this study, the serum concentrations
of C-reactive protein (CRP), haptoglobin (Hp), albumin, fibrinogen
and transferrin were investigated in dogs with spontaneous
hyperadrenocorticism (HAC) in order to establish a possible APP
profile for this disease. Materials: Acute phase proteins were
measured in the serum of three groups of dogs: healthy dogs
(n544), dogs with spontaneous HAC (n530), and dogs with spon-
taneous HAC and evident concurrent inflammatory conditions
(n512). Results: No significant differences for CRP and albumin
Vol. 35 / No. 4 / 2006 Veterinary Clinical Pathology
concentrations were found between healthy dogs and dogs with
spontaneous hyperadrenocorticism; however, significantly higher
values of Hp, fibrinogen and transferrin were found in the HAC
group. Dogs with spontaneous hyperadrenocorticism and evident
concurrent inflammatory conditions showed a significant increase
concentrations compared with the healthy animals group. Discus-
sion and Conclusions: In conclusion, a particular APP profile
induced by an excess of glucocorticoids has been identified in
patients with hyperadrenocorticism. However, this profile can be
significantly changed in the case of concurrent inflammation with
the appearance of a typical acute phase reaction.
11
PRESENCE OF DIROFILARIA REPENS LARVAE IN CYTOLOGI-
CAL SPECIMENS. P. Vajdovich, D. Szécsényi, D. Halmay.
Department of Internal Medicine, Faculty of Veterinary Science,
Szent István University, Budapest, Hungary.
Introduction: There is an increasing incidence of Dirofilaria repens
infestation in Hungary with endemic areas. Cytological specimens
from different organs of dogs may reveal microfilariae that are
mostly distinguishable from Dirofilaria immitis (heartworm). It
seemed to be important to evaluate whether these organisms cause
inflammatory, reactive or neoplastic conditions. Materials and
Methods: From 1995 until 2006 there were 22 cytological speci-
mens registered with the presence of Dirofilaria repens larvae. These
microfilariae were found in different numbers in the specimens.
A frequency analysis was performed according to various
parameters. Results: Mean age of dogs was 9.5 years (63.4), with
6 males and 6 females of different breeds. There is an increasing
incidence of dirofilariasis, as there were 1–4 cases until the end of
2004 and 11 cases were reported in 2005. With respect to seasonality,
cases were reported in spring (27%), in summer (14%), in autumn
(22%) and in winter (36%). Origins of cytological specimens were
skin nodules (n512) and different intrathoracic and intra-abdom-
inal areas (n510), such as pericardial cavity (n54), pleural effusion
(n51), liver mass (n53), and intra-abdominal cyst (n52). In
cytological samples 8 cases showed an inflammatory process
(24%), while in 14 samples (40%) reactive cell proliferation was
found. Degenerative changes of the liver were found in 3 samples
(9%). No reaction was found in 2 cases (6%) and in 7 samples
neoplastic conditions (21%) were reported. Discussion: The
relatively large proportion of cases with the presence of larvae in
neoplastic masses may raise a theory of an association between
parasitic infections and the development of tumours in dirofilariasis
similar to Spirocerca lupi infestations. Conclusion: In spite of the
low number of cases we can conclude that dominant alterations
caused by Dirofilaria repens are reactive rather than inflammatory
changes.
12
DETECTION OF CANINE DISTEMPER VIRUS BY LIGHT CY-
CLER REAL-TIME PCR IN DIFFERENT SAMPLES FROM ITAL-
IAN DOGS. M. Trotta, C. Zampieron, L.M. Solano-Gallego,
L. Razia, M. Caldin, T. Furlanello. Clinica Veterinaria Privata
‘‘San Marco’’, Padova, Italia.
Although canine distemper virus (CDV) is generally controlled with
live attenuated vaccines, natural CDV cases still occur in Europe. A
variety of assays have been developed for the definitive antemortem
diagnosis. However, there is a lack of diagnostic tests that permit
the differentiation of natural (N) and vaccine (V) CDV. The first
objective was to detect CDV in different tissues from suspected
Page 477
 ESVCP Annual Meeting Abstracts
sick dogs by Light Cycler PCR (LC-PCR) with Hybridization
probes (LC-PCR). The second objective was to determine if melting
curve temperature (Tm) could differentiate between natural
and vaccine CDV. From July 2003 to May 2006, a total of 587 sam-
ples (blood n5391, CSF n5113 and other tissues n583) were
tested. Five different commercial CDV vaccines were assessed. The
total prevalence and prevalence for blood, CSF and other tissues
were 21.8%, 26.9%, 8.8% and 15.6%, respectively. Similar Tm
were obtained from different CDV vaccines with a mean and SD
of 60.828C 6 0.83. The mean and SD of Tm of 58 CDV-positive
dogs was 57.798C 6 2.43 and was statistically significantly
lower than vaccine Tm (p50.0077). Positive dogs with Tm higher
than 58.38C were classified as V-like CDV (n519, 32%) and with Tm
lower than 58.38C were classified as N-like CDV (n539, 67%).
All dogs classified as N-like CDV were not or not recently vac-
cinated while dogs classified as V-like CDV were divided into
5 dogs with a recent CDV vaccine (less than 2 weeks) and 14 that
were not or not recently vaccinated. This study confirms the
usefulness of LC-PCR of different tissues for the diagnosis of CDV
infection in dogs. The results showed that N-like CDV is more
prevalent than V-like CDV. Natural infections were also observed to
belong to the V-like CDV group in unvaccinated dogs. Other
molecular techniques should be applied to differentiate between
wild type and vaccine CDV.
13
ASSESSMENT OF THYROID STATUS IN DAIRY COWS: SERUM
FREE AND TOTAL THYROXINE, IODINE AND SELENIUM. B.
Siliart, L. Martin, H. Dumonn. Endocrinology and Nutrition
Unit-LDH, National Veterinary School of Nantes, Nantes, France.
Introduction: Several studies on various troubles in dairy herds
have focused on Se and I deficiencies. As many other factors may be
involved in these diseases, it would be interesting to directly assess
thyroid status by measuring thyroxine, free (FT4) or total (TT4),
along with circulating iodine (I) and selenium (Se). Objective: To
establish reference values for serum FT4, TT4, I and Se in lactating
cows for routine control. Materials: Blood samples were collected
from the jugular vein prior to milking. Free thyroxine (FT4) and total
thyroxine (TT4) was assayed with RIA kits (Beckman-Coulter) and
iodine and selenium by ICP-MS. Animals: 122 healthy lactating and
pregnant Prim-Holstein cows, 3 to 5 years old from 11 different
herds, grazing on pasture during July in Brittany. Results: 1)
Means (m), standard deviations (sd), and percentiles (c) 5% and
95%: FT4 pM/L (m511.5, sd52.8, c555.9, c95517.5), TT4 nM/L
(m539.4, sd513.7, c5521.8, c95565.2), I lg/L (m582.1, sd 5 36.1,
c5541.7, c955178.4), Se lg/L (m555.7, sd513.2, c5534.5,
c95571.6). 2) FT4 was correlated with TT4 (p , 0.0001) but neither
FT4 nor TT4 were correlated with I (p50.46 and p50.23) or
selenium (p50.07 and p50.93). I was correlated with Se (p , 0.002).
3) Age and lactating number had no effect. The FT4 and TT4 values
were slightly higher in cows whose udders were cleaned with
iodinated solutions. A significant herd effect was found for all
values (p , 0.001). Discussion: The herd differences could be
related to the type of soil, forage and animal husbandry, but the data
were insufficient to perform a statistical analysis. Conclusion: The
reference serum values adopted by our laboratory for lactating
and pregnant cows are FT456 to 17.5 pM/L, TT4522 to 65 nM/L,
I542 to 180 lg/L, Se535 to 72 lg/L.
14
DEVELOPMENT OF SUBLINGUAL BLOOD SAMPLING IN MICE
WITH ASSESSMENT OF CLINICAL PATHOLOGY PARAMETERS
AND COMPARISON VERSUS RETROBULBAR SAMPLING. M.
Page 478 Veterinary Clinical Pathology
Heimann1, H.P. Käsermann2, R. Pfister1. 1Novartis Pharma AG,
Toxicology, Muttenz, Switzerland; and 2Institute of Laboratory
Animal Science, University of Zurich, Zurich, Switzerland.
The examination of blood is a key component in laboratory animal
studies. If large volumes of blood are required, the technique of
retrobulbar venous plexus puncture is still the method commonly
used for blood sampling in rodents. This technique is efficient but
has the potential to cause severe tissue damage. As an alternative
method a refined technique of sublingual blood sampling in rats
has been used since 1998. This method yields large volumes (up to 2
mL), does not cause severe tissue damage and, therefore, is the
recommended blood collection method in Switzerland today.
Objectives: Based on the effectiveness of this method in rats,
a study with anaesthetized male CD1 mice was conducted to
develop the puncture of the V. sublingualis in mice. Clinical
pathology parameters using this alternative technique versus the
conventional retrobulbar blood sampling method were compared.
Method: 300 lL of blood were collected from either the sublingual
vein or the retrobulbar venous plexus (n530 animals/group).
Hematology parameters—total and differential white blood cell
counts, red blood cells, hemoglobin, hematocrit, erythrocyte indices
and platelet counts—were determined using an ADVIA 120 (Bayer
Diagnostics, Munich, Germany). The clinical chemistry parame-
ters—alanine aminotransferase, albumin, alkaline phosphatase,
aspartate aminotransferase, glucose, triglyceride, urea, calcium,
chloride, potassium, sodium, and total protein—were determined
with a Synchron CX5 Beckmann Coulter. Results: Differences
between specific blood parameters were observed, depending on
the technique used. Increased values for leukocytes, lymphocytes
and monocytes were found in sublingual blood. Retrobulbar blood
yielded higher values for alanine aminotransferase, aspartate
aminotransferase and glucose. Conclusion: Sublingual blood
collection can be recommended as a suitable method in mice. Due
to the differences in hematology and clinical chemistry parameters,
the implementation of new reference values is recommended if
the new method is used.
15
FLOW CYTOMETRY TO DETECT PHOSPHOLIPIDOSIS IN
PERIPHERAL BLOOD LYMPHOCYTES OF RATS TREATED
WITH AMIODARONE. M.E. Perico, F. Crivellente, P. Cristo-
fori. Clinical and Molecular Pathology Laboratory, Safety Assess-
ment Department, GSK Research Centre, Verona, Italy.
Introduction: A flow cytometry method to monitor peripheral
lymphocyte phospholipidosis has been set up using double-staining
with Nile Red and anti-CD3 monoclonal antibody (MAb). Blood
was collected from rats treated with amiodarone (antiarhythmic
drug, phospholipidogenic). By flow cytometry, it is possible to
detect neutral lipids (NL) and phospholipids (PL) using Nile Red,
a probe for intra-cellular lipid staining, changing its fluorescence on
the stained lipid basis. CD3 antigen was selected to focus on T cells,
to evaluate if these cells are the target of amiodarone-dependant
phospholipidosis. Materials: Sprague-Dawley male rats were
treated for 14 days with vehicle or 300 mg/kg/day amiodarone
(n55). Peripheral Blood Mononuclear Cells (PBMC) were separated
from whole blood by centrifugation on a density gradient. Cells
were first stained with anti-CD3, then fixed with 1% paraformal-
dehyde and finally stained with Nile Red. Samples were acquired
and analysed using a Partec PAS III flow cytometer. Results: CD3
positive T lymphocytes (CD3þ) represent 40%, in both controls and
treated rats. In the total lymphocyte population, PLþ positive cells
are 1% for controls animal, and more than 5-fold higher (about 6%)
for treated animals (p 5 0.008). Focusing only on CD3þ cells, the
Vol. 35 / No. 4 / 2006
 ESVCP Annual Meeting Abstracts
difference is more significant: PLþ cells are 10% for controls and
40% for treated animals (p 5 0.0056). As expected, there are no
evident changes of CD3þ NLþ cells. Comments: Double-staining
with MAb and Nile Red gives very good results and it allows
focusing on specific cell populations. Results show that amiodar-
one-treated animals significantly increase the number of vacuolated
lymphocytes (PLþ). The phospholipidogenic effect is more evident
focusing on CD3þ lymphocytes, thus suggesting that these cells are
probably the target of phospholipidosis.
16
HEPATOPROTECTIVE EFFECT OF CHICORY (CHICORIUM IN-
TYBUS) IN BROILERS. P. Khazraii Nia, M. Torabi Goodarzi, M.
Modirsanei, P. Yussefi. Department of Clinical Sciences, Faculty
of Veterinary Medicine, University of Tehran, Tehran, Iran.
It has been proved that Chicory root stimulates the production of
bile and empties the gall bladder. This study was conducted on the
hepatoprotective effect of Chicory on broilers with experimental
hepatotoxicity. One hundred twenty (120) one-day-old chickens
(approximately the same weight) were chosen. They were divided
randomly into three groups of 40 chickens each. All of them were
fed a normal diet for one week and on the second week group one
were fed a normal diet (the control group). The second and third
groups received tetracycline (4 gr/liter of water). Blood samples
were collected on the 15th day. In the third week group one was
continued on a normal diet (control group) and the second and third
groups (tetracycline groups) had 2% and 1% Chicory powder,
respectively, added to their diet. Blood samples were collected on
day 21. ALT, AST, LDH and GGT serum activities were measured on
serum samples from the 15th and 21st days. The mean serum GGT
activity on the 15th day for groups two and three (tetracycline
groups) was significantly increased compared with 15-day-old
control group chickens. The maximum and minimum serum AST
activities were shown in group two and three 15-day-old chickens
(tetracycline) and group two 21-day-old chickens (2% Chicory
powder added to their diet after tetracycline), respectively. There
was a significant decrease in the mean of serum GGT activity in
chickens fed 2% Chicory compared with chickens that received an
overdosage of tetracycline during the 2nd week. There were no
significant differences in ALT and LDH serum activity between
15th day sera (control and treatment) and between sera of chickens
that received an overdosage of tetracycline and chickens that
received Chicory in 3rd week. The result of this study suggests
that adding 2% Chicory powder has hepatoprotective effects on
liver toxicity due to overdosage of tetracycline in broilers.
17
VARIATION IN PLASMA ESTRADIOL AND PROGESTERONE
CONCENTRATION IN PREGNANT MARES: APPLICATION
FOR A ROUTINE TEST OF GESTATION. B. Siliart, L. Martin,
H. Dumon. Endocrinology and Nutrition Unit, LDH, National
Veterinary School of Nantes, Nantes, France.
Introduction: Levels of the sex hormones, estradiol and pro-
gesterone, show important variations during pregnancy. Whereas
the reproduction specialist can use trans-rectal echography to
follow-up gestation, a laboratory test is required for non-specialist
veterinarians, or for dangerous mares. Objective: To establish
reference values for estradiol and progesterone in the mare for
routine laboratory diagnosis of gestation. Material and methods:
Estradiol and progesterone were assayed in plasma. Blood samples
were collected from the jugular vein, centrifuged and decanted and
Vol. 35 / No. 4 / 2006 Veterinary Clinical Pathology
the serum frozen (
208C) until assayed. Estradiol was assayed with
a RIA kit from Diasorin (ref P2210) and progesterone with the RIA
kit from Immunotech (ref 1188). All assays had been validated for
horses. Case records of pregnant mares, referred to our laboratory
for hormonal assay and with known date of insemination, were
reviewed. Results: 520 mares were included in the study.
Hormone concentrations varied throughout gestation, from 19 (2)
to 921 (181) pM with a peak at the 9th month for estradiol and from
15 (2) to 16 (4) nM with a peak at the 4th month for progesterone.
The sensitivity of the estradiol assay used as a routine test to
diagnose gestation was 74% after the 5th month (threshold value
100 pM) and .95% after the 6th month (threshold value 150 pM).
The progesterone assay was the best routine test for earlier
diagnosis (threshold value: 8 nM). The sensitivity then varied from
83% (month 2) to 67% (month 5). Sensitivity could be increased to
95.4% throughout gestation by taking the 2 hormones into account
together with respective threshold values of 8 nM and 150 pM.
Conclusion: These results show that estradiol and progesterone
assays are useful for the routine biological test of gestation in mares
after an appropriate choice of threshold values.
18
THE OVINE ACUTE PHASE RESPONSE TO VACCINATION IN
RELATION TO NUTRITION DURING GESTATION. P.D. Eck-
ersall1, F. Lawson1, C.E. Kyle2, M. Waterston1, F. Campbell1,
S.M. Rhind2. 1Division of Animal Production and Public Health,
Faculty of Veterinary Medicine, University of Glasgow, Glasgow,
UK; and 2Macaulay Institute, Craigiebuckler, Aberdeen, UK.
The acute phase response is the immediate host response to
infection, inflammation and trauma and can be monitored by mea-
suring proteins (APP) such as haptoglobin (Hp) or serum amyloid
A (SAA). The plane of nutrition during pregnancy is known to
affect the resistance to disease of neonatal animals but effects on
the resting concentration of APP and the dynamics of their re-
sponse following stimulation are unknown. Here, the non-stim-
ulated serum concentration of Hp and SAA was determined in
lambs from 3 groups (n510/group). The dams of the lambs of the
respective groups were fed 65% of live weight maintenance
requirements for the first 65 d gestation followed by 100% of
requirements (increasing according to fetal burden) for the re-
mainder of pregnancy (Low/High; LH); 100% of requirements for
the first 65 d of gestation followed by 70% of requirements until 125
d when they were fed 100% of requirements to ensure adequate milk
production (High/Low; HL) or 100% of requirements throughout
gestation (High/High; H/H). The dynamic acute phase response
in the lambs was estimated by measuring the concentration of the
APP following routine vaccination with Heptavac (Intervet)
between days 8–14 and at re-vaccination between days 43–49 after
birth. Concentrations of APP were determined before each
vaccination and at 1, 2, 3, 4, 6 and 8 days post-vaccination. There
was no significant difference between groups in the non-stimulated
concentrations of Hp or SAA. Vaccination stimulated the acute
phase response, as measured by area under the Hp and SAA curves
(AUC), and there were reductions in the response of Hp and SAA
to vaccination in lambs born to ewes undernourished for part of
gestation but the differences were not significant. The mean AUC of
the SAA response was greater after the 1st than the 2nd vaccination
(P , 0.001) while there was no such difference in the equivalent
Hp response. While no effect of maternal nutrition was found, the
ability to monitor the dynamic acute phase response following
vaccination may be a valuable procedure to assess individual
differences in their host responses to infection.
Page 479
 ESVCP Annual Meeting Abstracts
19
ACUTE PHASE PROTEINS IN THE DIAGNOSIS OF BOVINE
SUBCLINICAL MASTITIS. S. Safi1, A. Khoshvaghti1, S.R.
Jafarzadeh2, T. Taghvai3. 1Department of Clinical Pathology,
Faculty of Specialized Veterinary Medicine, Islamic Azad Univer-
sity, Science and Research Branch, Tehran, Iran; 2Department of
Medicine and Epidemiology, School of Veterinary Medicine,
University of California, Davis, CA, USA; and 3Faculty of Veterinary
Medicine, University of Tehran, Tehran, Iran.
Introduction: Economic loss due to subclinical mastitis is much
greater than that from acute mastitis because the reduction in milk
production is gradual, persists for long periods of time, and
undermines the yield of the infected cow. For every clinical case of
mastitis, there will be 15 to 40 subclinical cases. Subclinical mastitis
is mostly diagnosed by cow-side tests such as the California Mastitis
Test (CMT), or by laboratory analyses of somatic cell count (SCC)
using automatic cell counters. It has been suggested that neither
CMT score nor SCC is sensitive enough to be useful as a screening
test for identifying infected mammary quarters among dairy cattle;
therefore, it is of importance to investigate alternative methods for
more objective and early diagnosis of subclinical mastitis. Acute
phase proteins (APR) are a group of proteins which undergo
substantial changes in concentration following infection, inflamma-
tion or trauma. There are few reports on APR during episodes of
subclinical mastitis. The objective of this study was to evaluate and
compare the accuracy of APR with CMT and SCC in the diagnosis of
subclinical mastitis in dairy cows. Materials and Methods: Indi-
vidual quarter milk from 173 Holstein cows on 7 dairy farms around
Shiraz, Iran were sampled. The cows were milked three times daily.
California Mastitis Test (CMT) and automated counting of somatic
cells (SCC) were performed on milk samples from each quarter.
Milk samples were submitted for routine bacteriological examina-
tion. The concentrations of haptoglobin in milk (MHP) and amyloid
A in milk (MAA) and serum (SAA) were determined using ELISA
kits (Tridelta Development Ltd, Ireland) and the concentration of
haptoglobin in serum was determined using a Haptoglobin Kit
(Tridelta Development Ltd, Ireland). Receiver-operating character-
istic (ROC) analyses were performed to assess the overall accuracy
of CMT, SCC, MAA, MHP, SAA, and SHP, and to select cut-off
points which provide about 90% sensitivity in diagnosis of
subclinical mastitis using bacterial culture as a gold standard. The
areas under the curves (AUC) of each pair of tests were compared
using Pearson’s Chi-square test. Results: The bacteriological and
SCC results from the subclinical cases of mastitis and from the
healthy cows showed a significant difference (P , 0.005). MAA,
CMT, and SCC had the highest overall accuracy for diagnosis of
subclinical mastitis and were not significantly different from each
other, but had significantly higher overall accuracy than MHP, SAA,
and SHP. Suggested cut-offs for CMT, SCC, MAA, MHP, SAA, and
SHP were 0, 130.0 3 103, 16.40 mg/ml, 3.90 ng/ml, 159.10 mg/ml,
and 0.05 mg/ml, respectively. Sensitivities (95% CI) of the above
mentioned parameters were 100, 89.6, 90.6, 90.0, and 90; specificities
(95% CI) were 77.1, 72.0, 98.3, 68.6, 72.1, and 43.0 and AUC (95% CI)
were 0.965, 0.948, 0.998, 0.886, 0.863, and 0.62, respectively.
Discussion: The significant difference between milk haptoglobin
and amyloid A in healthy cows and cows with subclinical mastitis
indicated that an acute phase response had occurred and that these
proteins may have the potential to be used as diagnostic tests for
the diagnosis of subclinical disease. ROC analysis provides an
independent way of evaluation of continuous or ordinal tests in
clinical pathology laboratories, and the AUC gives an overall
measure of test accuracy. We proposed cut-off points for CMT, SCC,
MAA, MHP, SAA, and SHP in diagnosis of subclinical mastitis with
high sensitivity (about 90%). While our results suggested MAA as
the most accurate test in this regard, the AUCs of CMT and SCC
were also significantly higher than MHP, SAA and SHP which
Page 480 Veterinary Clinical Pathology
suggests that tests on milk generally provide more accuracy than
tests on serum in the diagnosis of subclinical mastitis.
20
CYSTATIN C AS AN INDICATOR OF RENAL FUNCTION IN
DOGS. E. Lock, N. Bexfield, J. Archer. Department of Veterinary
Medicine, University of Cambridge, Cambridge, UK.
Cystatin C is a small peptide which acts as a protease inhibitor
and is produced by all nucleated cells, filtered by the glomerulus
and reabsorbed in the proximal tubules. In human medicine it is
used as an endogenous marker of renal function, and can be used
to estimate glomerular filtration rate (GFR) based on a specific
formula. It has been proposed that this formula may be used as an
alternative to the estimation of GFR by plasma clearance methods.
A particle-enhanced turbidimetric immunoassay (PETIA) designed
for measuring cystatin C in human plasma and serum has been
validated for use in dogs (Jensen AL, Bomholt M, Moe L. Vet Clin
Pathol. 30:86–90, 2001; Almy FS, Christopher MM, King DP, Brown
SA. J Vet Int Med. 16:45–51, 2002). Because a decline in GFR with age
has been demonstrated for humans, a parallel study of GFR in
healthy dogs using the plasma clearance of iohexol was established
by one of us (N Bexfield). Samples from these dogs, grouped
according to age, were used for cystatin C determination and
establishment of age-specific reference intervals. Measurements of
cystatin C were also made on serum from dogs with established
renal disease for evaluation of this method as a marker of renal
compromise. The assay measured canine cystatin C in a linear and
proportional manner with a mean recovery of 109.1% 6 7.1%, with
a CV of 3.3%–10.6%. Haemolysis at a concentration .0.1 g/L was
shown to affect the assay. The ‘normal’ group of 101 dogs was
subdivided according to age into groups with age ranges 0–3, 4–6,
7–9 and 10þ years old. The reference limits established for cystatin
C were 0–3 years, 0.36–0.72 mg/L; 4–6 years, 0.34–0.75 mg/L; 7–9
years, 0.38–0.76 mg/L; and 10þ years, 0.26–0.74 mg/L. There was
no significant difference between genders or age groups for healthy
animals. The mean cystatin C levels were significantly higher in
a group of 49 azotaemic dogs with serum urea .8.0 mm/L and
creatinine .150 lm/L than in clinically healthy ‘normal’ dogs in all
age ranges at p , 0.01. Azotaemic dogs had serum cystatin C
levels ranging between 0.84
1.22 mg/L. Cystatin C levels and
GFR measured by iohexol clearance in these dogs need to be
correlated and larger numbers of dogs with severe azotaemia and
dogs with normal creatinine levels but increased GFR need to be
studied.
Poster Presentations
21
COMPARATIVE STUDY ON IRON DEFICIENCY ANEMIA IN
PUPPIES FED MATERNAL AND NON-MATERNAL MILK. S.J.
Aldavood, P. Keyhani, A.R. Karegar, A. Moghadami, H.
Bahrami Gorgi. Azad University, Science and Research Center
Campus, Shahrekord Azad University, Veterinary Faculty, Iran.
Anemia and particularly iron deficiency can affect the growing
process negatively. Anemia is one of the most important problems
in growing puppies. It can have a lot of negative effects on the
growing process. Iron deficiency anemia is produced by depletion
of iron reserves. Due to this depletion, no iron is available for
haemoglobin (Hb) synthesis. Iron reserve depletion happens when
Vol. 35 / No. 4 / 2006
 ESVCP Annual Meeting Abstracts
the iron consumption or iron loss is considerably more than
synthesis. In this study 2 groups of puppies (25 puppies in each
group) an average of 3 months old were randomly selected. The first
group was considered as the control group and the puppies of this
group were fed maternal milk. The second group was the test group
and the puppies of this group were separated from their mother
at two weeks of age. These puppies were fed manually with the
cow’s milk. One ml of blood was collected from each puppy and
haematological factors such as red blood cell count (RBC),
haemoglobin concentration (Hb), hematocrit (PCV) and cellular
indexes of MCHC, MCH and MCV were measured. The results
showed that there is a significant difference in ‘‘mean red blood cell
count’’ between control and test groups (P , 0.05). Differences in
‘‘MCHC’’ between these two groups were also significant (P , 0.05).
Moreover, all of these parameters were compared statistically with
their corresponding standard values and all differences were
significant except for ‘‘MCHC’’ and ‘‘MCV’’ between the control
group and relevant standard values. This means that the RBC in
puppies fed with maternal milk are normocytic-normochromic and
somehow iron deficiency in this group of puppies can be ruled out.
On the other hand, ‘‘MCV’’ in the test group was significantly
different to normal values, so in this group the RBC are microcytic-
hypochromic and this issue can be an early stage of iron deficiency
anemia.
22
CYTOLOGIC FEATURES OF RETROPERITONEAL MASSES IN
DOGS—SOMETIMES TRICKY! A. Guija de Arespacochago1, K.
Hittmair2, S. Shibly3, I. Schwendenwein1. 1Central Laboratory,
2
Clinic for Diagnostic Imaging, 3Institute of Pathology and Forensic
Veterinary Medicine, University of Veterinary Medicine, Vienna,
Austria.
Ultrasound-guided fine needle aspiration biopsy (FNAB) of internal
organs usually allows a timely diagnosis and the establishment of
an adequate therapeutic plan. The accuracy of cytology regarding
the cytology of internal organs is close to 80% compared with
histology. Identification of the tissue of origin can be very difficult in
cases where diagnostic imaging techniques fail to localize the lesion.
In a 2-year period 7 cases of retroperitoneal masses were sampled:
6 ultrasound-guided FNAB and one intra-operative touch im-
print. A histological diagnosis has been established in 6/7 tu-
mours. Cytological features of adrenal gland adenocarcinoma
(n53), granulosa cell tumour (n52), and anal sac adenocarcinoma
(n52) were compared. Anal sac adenocarcinomas and granulosa
cell tumours showed similar cytologic features, such as high cellular-
ity, mild anisokaryosis, acinar arrangement of cells, some naked
nuclei, indistinct cell borders, small amounts of cytoplasm, fine chro-
matin pattern and indistinct nucleoli. Cytological features of adrenal
gland adenocarcinomas were the most inconstant: one showed
features of naked nuclei and fatty vacuoles in the background
considered characteristic for neuroendocrine tumours; another one
was misdiagnosed as granulosa cell tumour because ultrasound
examination suspected ovarian tissue. Cytologically there was
a mixed population of naked nuclei, round cells and cells with an
acinar arrangement, and an intermediate grade of anisokaryosis
mixed with some giant nuclei with prominent nucleoli. The third
case, a ruptured tumour, showed only few monomorphic slightly
polygonal cells with indistinct cell borders. Cytological differenti-
ation between anal sac adenocarcinomas and granulosa cell tu-
mours was difficult. The presence or absence of a perianal tumour
combined with a retroperitoneal mass in an intact bitch might help
identify the tissue of origin. In adrenal gland carcinoma without
typical features for this tumour, marked anisokaryosis could help to
distinguish this from the other two. In cases of atypical cytologic
Vol. 35 / No. 4 / 2006 Veterinary Clinical Pathology
features when exact localization is not available, determination of
histogenesis by cytology alone may be impossible.
23
A RETROSPECTIVE STUDY OF 82 CASES OF CANINE LYM-
PHOMA IN AUSTRIA USING THE WORKING FORMULATION
AND IMMUNOPHENOTYPING. A. Guija de Arespacochaga1,
I. Schwendenwein1, H. Weissenböck2. 1Central Laboratory,
2
Institute of Pathology and Forensic Veterinary Medicine, Univer-
sity of Veterinary Medicine, Vienna, Austria.
In humans, numerous studies have shown that the prevalence
of different non-Hodgkin’s Lymphoma (NHL) subtypes differs
strongly from region to region. A similar pattern is assumed to exist
in dogs, but not enough empiric research has been conducted yet.
The aim of this study was to classify canine lymphomas in Austria
according to their morphology and phenotype and to compare the
results to the findings of similar studies in other countries. We
classified 82 NHL according to their morphology based on the
Working Formulation and their phenotype was determined by
using anti T-cell and anti B-cell antibodies. Out of these, 42 (51.2%)
were of B-cell subtype, 24 (29.3%) of T-cell subtype, and 16 (19.5%)
remained unclassified, either because they showed a double
staining of the cells (4/16) or a negative staining (12/16). Diffuse
lymphomas predominated (99%) over follicular lymphomas, while
intermediate grade lymphomas (61%) outnumbered high grade
lymphomas (23.2%) and low grade lymphomas (13.4%). The most
common subtype was the diffuse large cell lymphoma (40.2%),
followed by the large cell immunoblastic lymphoma (13.4%) and the
diffuse small lymphocytic lymphoma (13.4%), while follicular large
cell lymphoma and small noncleaved cell lymphoma were un-
derrepresented at 1.2%. Generally, these results are in line with the
findings of similar studies in Western Europe, and only small
differences concerning some subtypes were found. Some of
these differences could be caused by different classification ap-
proaches, differences in antibody specificities or the low number of
patients included in the studies. The remaining, minor dif-
ferences could be caused by geographic factors, but the overall
similarity of the results to the findings of former studies in Western
Europe makes the existence of specific risk factors in Austria
rather unlikely.
24
CHRONIC MYELOPROLIFERATIVE DISORDER IN A FOUR-
YEAR-OLD PERSIAN CAT. N. Atyab1, N. Korrami2, R. Maza-
herinezhad3. 1Department of Clinical Science, College of
Veterinary Medicine, University of Tehran, Tehran, Iran; and 2Small
Animal Internal Medicine and 3Clinical Pathology Laboratory,
College of Veterinary Medicine, University of Tennessee, Knoxville,
TN, USA.
A 4-year-old Persian cat was presented to the small animal hospital
at the college of veterinary medicine. The animal was recumbent,
depressed, and dehydrated, with anorexia and complications in
breathing. The owner complained about diarrhoea and vomiting
during the last two days. The cat’s oral mucosal membranes and
gingiva were pale and a little icteric. The haemogram showed
PCV533%, WBC5100,000/ll, segmented neutrophil572%, band
neutrophil57%, lymphocyte510%, eosinophil58% and basophil5
7%. There was severe proliferation of the myeloid series and
presence of a neutrophilic left shift and toxic neutrophils. The
erythroid series proliferation was also severe with 46% nucleated
red blood cell (NRBC) and the presence of blast forms of RBCs.
Rouleaux formation was noticed in the erythrocytes and there were
Page 481
 ESVCP Annual Meeting Abstracts
signs of anaemia with the presence of hypochromia and Howell-
Jolly and Heinz bodies. The packed cell volume (PCV) seemed,
however, in normal range, but the anaemia and high serum protein
level (7.9 g/dl) suggested it should be below the normal range. The
liver enzymes showed high elevation in their activities, (AST . 130
U/L and ALT . 130 U/L) which suggested hepatic dysfunction.
There were inflammatory processes in the liver, lungs and intestine,
and the liver was larger than normal on the radiographs. There was
no sign of splenomegaly on the radiographs. The patient did not
respond to therapy and died several days after treatment. A
myeloproliferative disorder was diagnosed with erythroleukemic
reaction, predominanantly MDS-Er or M-Er, in which the GI tract
was involved and produced diarrhea and vomiting. The other
organs like lungs and liver were involved due to cellular infiltration.
25
EVALUATION OF BOVINE COLOSTRAL IgG AND TOTAL
PROTEIN AND THEIR RELATION TO THE ENZYMES GGT
AND ALP. N. Atyabi, F. Gharagozloo, P. Khazraiinia, A.
Bahonar, M. Taheri. Department of Clinical Science, College of
Veterinary Medicine, University of Tehran, Tehran, Iran.
A survey was conducted to investigate the quality of bovine
colostrum, absorption of its immune proteins and the resulting
passive immunity transferred to newborn calves. Total protein (TP),
immunoglobulin G (IgG), and the enzymes gammaglutamyl trans-
ferase (GGT) and alkaline phosphatase (ALP) were assayed, em-
ploying 65 bovine colostrum samples. Samples were taken after
parturition. Immunoglobulin G (IgG) was assayed by an ELISA
method. The assayed parameters were as follows: total protein 17.75
6 0.79 g/dl, IgG 12145.09 6 822.96 mg/dl, GGT 10211.17 6 279.52
U/L and ALP 2243.69 6 188.13 U/L. The resulting data revealed
huge amounts of TP and IgG in colostrum pointing to the fact that
there is a significant correlation among these two parameters. A
significant correlation was found between ALP and IgG. The same
correlation was also seen between GGT and IgG. The resulting data
show the colostrum total protein, and the enzymes GGT and ALP
which are potentially increased with production of colostrum, could
help to evaluate the quality of colostrum and if it could transfer
sufficient passive immunity to newborn calves.
26
IRON SUPPLEMENTS ARE REQUIRED FOR SUCKLING
CALVES. N. Atyabi, F. Gharagozloo, S.M. Nassiri. Department
of Clinical Sciences, School of Veterinary Medicine, University of
Tehran, Tehran, Iran.
Iron has been touted as a necessary element for the normal growth
of newborn calves. On the other hand, milk is considered to be
a poor source of iron. To exactly investigate the iron requirements of
Holstein calves during different periods of growth, we designed this
study. Serum iron (Fe), total iron binding capacity (TIBC), cerulo-
plasmin and ferritin were measured in 46 newborn calves. Blood
samples were collected from calves at four distinct times: before
suckling colostrum (T0), after suckling at one-day-old (T24), two-
day-old (T48) and two-month-old (T2mon). Blood iron levels in
newborn calves, before suckling colostrum, were the same as the
mothers’, but decreased in one-day-old and two-day-old calves and
continued to decrease until two-months-old. There were significant
differences between serum iron level at T0 and T48 and T2mon (P ,
0.001). Blood TIBC level was significantly higher in newborn calves
at T0 than their mothers’ (P , 0.001), then continued to significantly
increase in T24, T48 and T2mon (P , 0.001). Ceruloplasmin at
T2mon was in the normal range of the normal adult cow. That
means transport of iron in two-month-old calves, at least in that part
which is related to blood ceruloplasmin, is normal. Blood ferritin
Page 482 Veterinary Clinical Pathology
levels in newborn calves, before suckling colostrum, were the same
as their mothers’, but decreased in T24 and T48 calves, then
insignificantly increased to two-months-old. The data showed that
suckling calves require iron supplements. These are essential for
calves at birth and may play an effective role in their growth,
hematopoiesis and resistance to infections.
27
USE OF EDTA SAMPLES FOR HEMOSTASIS TESTING. J.J.
Ceron1, E. Carli2, S. Tasca2, S. Martinez-Subiela1, C. Patron2,
M. Caldin3. 1Animal Medicine and Surgery Department, Faculty of
Veterinary Medicine, Murcia, Spain; 2Laboratorio dAnalisi Veter-
inarie ‘‘San Marco’’, Padova, Italy; and 3Clinica Veterinaria Privata
San Marco, Padova, Italy.
Introduction: Tests for coagulation are usually made on blood
collected with citrate tubes. However, EDTA is recommended for
CBCs and other hematological analysis, and the possible use of the
same EDTA sample for performing hemostasis tests would have
evident advantages in routine practice. The purpose of this study
was to evaluate the use of EDTA tubes for the analysis of different
parameters of hemostasis such as prothrombin time (PT), activated
partial thromboplastin time (aPTT), fibrinogen concentration,
antithrombin (AT) activity and D-dimer concentration in dogs.
Materials: Blood was collected from 50 dogs and placed in EDTA
and citrate commercial tubes. In each paired plasma sample PT,
aPTT, and fibrinogen concentration were measured on an auto-
mated coagulation analyzer with commercially available reagents
and AT activity and D-dimer concentration were measured on an
automated chemistry analyzer using validated kits. Results: The
regression equations for the different parameters from citrate (y)
and EDTA (x) plasma were: PT y51.268x þ 0.063, R50.90; aPTT y 5
0.838x þ 1.934, R50.95; fibrinogen concentration y50.759x þ 63.6,
R50.97; AT y51.125x þ 2.402, R50.98 and D-dimer y51.110x þ
0.018, R50.99. Discussion and conclusions: A good correlation
(higher than 0.90) was observed for the values of all parameters
tested with citrate and EDTA. With the establishment of appropriate
reference range values, EDTA samples could be used for hemostasis
testing as an alternative to citrate. This would allow the use of only
one sample tube for performing hematology and coagulation tests
in routine practice.
28
COMPARISON OF DOG EOSINOPHIL COUNTS BY STANDARD
BLOOD SMEAR ANALYSIS AND PHLOXINE B STAIN WITH
RESULTS OBTAINED WITH THE LASERCYTE AND HEMAVET
950 ANALYSERS. J. Coillard, C. Lafond, P. Murgier, N.
Bourges-Abella, C. Trumel, J.P. Braun. Departement des
Sciences Cliniques, Service de Medecine des Carnivores Domes-
tiques, Ecole Nationale Veterinaire, Toulouse, France.
Introduction and Background: Eosinophilia is an uncommon
haematological change in dogs but has high medical significance in
various diseases. Most blood analysers have not been validated for
eosinophil counting in animals. This study was designed to
compare eosinophil counts by blood smear analysis as a reference
to results of different methods of analysis. Materials: Eosinophil
counts were performed on 58 EDTA-K3 canine blood samples by
manual counts on blood smears and manual counts after phloxine B
1% stain, and by Hemavet 950 (impedance technology, Drew
Scientific, UK) and LaserCyte (flow cytometry technology, Idexx
Laboratories, USA) analysers. Results were compared by Passing-
Bablok’s regression procedure and Bland Altman plot (Analyse-it,
Leeds, UK). Results: Correlation agreement between phloxine (y)
and blood smear (x) was good for most values: r50.789 and
Vol. 35 / No. 4 / 2006
 ESVCP Annual Meeting Abstracts
Passing-Bablok’s equation [95% CI]: y50.918 [0.775; 1.115]x þ 0.02
[0.005; 0.041]. Correlation agreements between the manual methods
and the Hemavet 950 were also good (Hemavet (y) vs. blood smear
(x): r50.74 and y50.934 [0.730; 1.157]x þ 0.069 [0.030; 0.083];
Hemavet (y) vs. phloxine (x): r50.76 and y51.179 [0.913; 1.541]x þ
0.013 [
0.048; 0.047]), whereas they were poor with the LaserCyte
(LaserCyte (y) vs. blood smear (x): r50.37 and y50.459 [0.228;
0.652]x þ 0.050 [0.030; 0.067]; LaserCyte (y) vs. phloxine (x): r50.45
and y50.585 [0.353; 0.800]x þ 0.034 [
0.016; 0.069]). Discussion &
Conclusion: In this study, no case of canine eosinophilia (GNE .
1.5 3 10 9 L–1) was detected and all methods agreed on this point.
Dog eosinophils are easily counted on standard blood smears.
Phloxine B stain could be a new method for total eosinophil
counting. Impedance technology of Hemavet 950 seems to correctly
distinguish dog eosinophils from the other leukocytes, whereas
flow cytometry of LaserCyte does not. However, this study must be
completed by analysis of canine blood samples with eosinophilia.
29
FUNGAL PYELONEPHRITIS DUE TO CLADOPHIALOPHORA
BANTIANA IN A DOMESTIC CAT CONTINUOUSLY RESIDENT
IN THE UNITED KINGDOM. O. Coldrick1, C.L. Brannon1,
D.M. Kydd2, G. Pierce-Roberts2, A.M. Borman3, A.G. Tor-
rance1. 1Torrance Diamond Diagnostic Services, Unit G, The
Innovation Centre, University of Exeter, Exeter UK; 2Kydd & Kydd
Veterinary Health Clinic, London, UK; and 3Mycology Reference
Laboratory, Southwest Health Protection Agency, Bristol, UK.
An eleven-year-old domestic shorthaired cat, continuously resident
in the United Kingdom, presented with weight loss, anorexia and
pyrexia. The left kidney was enlarged and painful on palpation
and ultrasonography revealed a grossly dilated renal pelvis from
which 13 mls of thick flocculent fluid were aspirated. Cytological
examination of this material revealed pyogranulomatous inflam-
mation. No bacteria were seen but there were numerous fungal
hyphae associated with adherent leukocytes. Similar fungal hyphae
were detected in the urine. The aspirated material was cultured on
Sabouraud’s Dextrose Agar at 308C and after 14 days small dark
green/olivaceous fungal colonies were detected. Bacterial culture
was negative. The preliminary identification of Cladophialophora
bantiana (based on colonial characteristics, morphology of hyphae
and conidia and an ability to grow at 428C) was confirmed by
sequencing PCR products corresponding to the large ribosomal
subunit region of the nuclear ribosomal repeat. These sequences
were identical to those of known isolates of C. bantiana maintained
at the National Collection of Pathogenic Fungi. Nephrectomy was
declined. Itraconazole therapy (Itrafungol; Janssen) was instituted
at 5 mg/kg once daily by mouth. Despite an initial six weeks of
clinical improvement the cat was euthanised due to suspected
involvement of the right kidney and deteriorating renal function.
C. bantiana is a dematiaceous mould present in soil and rotten
plant material, often in tropical or sub-tropical regions. Infection
with the pigmented hyphae of dematiaceous moulds is referred to
as haeohyphomycosis, of which C. bantiana is an infrequent cause in
human and animal patients. C. bantiana is, however, neurotropic in
both immunocompromised and healthy humans and causes brain
abscesses which are often fatal. Cutaneous and systemic cases are
also reported. CNS and, less frequently, systemic and cutaneous
cases have been reported in domestic cats and dogs. In most
cases, as in this case, no evidence for immunocompromise has been
found. Recently, three feline cases have been reported in Europe.
To the best of our knowledge this is the first case of infection with
C. bantiana in human or animal patients continuously resident in
the United Kingdom.
Vol. 35 / No. 4 / 2006 Veterinary Clinical Pathology
30
CAPILLARY ELECTROPHORESIS AS A VALUABLE TOOL TO
DETECT BILIRUBIN IN RAT SERUM. F. Crivellente, N.
Bocchini, L. Vandin, P. Cristofori. Clinical and Molecular
Pathology Laboratory, Safety Assessment Department, GSK Re-
search Centre, Verona, Italy.
Introduction: Bilirubin is the main bile pigment that is formed
from the breakdown of heme in red blood cells. The broken down
heme travels to the liver, where it is secreted into the bile by the liver.
A small amount of bilirubin circulates in the blood and serum
bilirubin is considered a true test of liver function. Background: In
drug pre-clinical development, the rat represents the rodent species
used to assess the toxicity of new chemical entities. Bilirubin
determination in rat serum is routinely carried out by means of
colorimetric methods on automatic biochemistry analyzers. Ob-
jective: The colorimetric methods currently used to measure
bilirubin are sometimes a cause of false positive results due to
haemoglobin contamination of serum (haemolytic serum) or direct
interferences caused by the chemistry of some compounds. For this
reason, capillary electrophoresis (CE) was evaluated as an alterna-
tive tool to detect bilirubin in rat sera because the complete
automation, the low amount of sample required, and low costs of
analysis make CE a valuable alternative tool. Materials: A
Beckman P/ACE MDQ instrument equipped with an UV-detector
has been used with different analytical conditions to identify
bilirubin in rat serum samples and to compare the results obtained
with the colorimetric method currently used. Results and
Discussion: It has been demonstrated that the present CE method
is capable of detecting bilirubin in rat serum samples with
reproducible results and good sensitivity. Abnormal high bilirubin
levels due to the presence of haemoglobin or attributable to the
chemistry of specific compounds in the serum can be easily
distinguished by means of CE. Conclusion: The method of CE
for determination of bilirubin in rat serum represents a good
alternative method in the identification of interferences due to the
presence of haemoglobin or of specific compounds.
31
COMPARATIVE HISTOPATHOLOGICAL CHANGES IN LIVERS
OF BULLOCKS SLAUGHTERED IN SALINE AND NORMAL
WATER AREAS OF THE WESTERN VIDHARBHA REGION OF
INDIA. P.S. Gandhewar, P.S. Deshmukh, V.P. Pathak, D.H.
Rekhate, M.V. Joshi. Department of Veterinary Pathology, Post
Graduate Institute of Veterinary and Animal Sciences, Akola,
Maharastra Animal and Fishery Sciences University, Nagpur,
Maharastra, India.
The Purna river valley of the western Vidarbha region is a unique
location in the world where the underground water up to 25 km
area on both sides of the Purna River is known to have very high
water salinity (above 6000 ppm). With a view to investigate the
impact of water salinity present studies were undertaken by
collecting 120 liver tissues each from bullocks slaughtered in a saline
water area (SWA), i.e., the Daryapur slaughterhouse and a normal
water area (NWA), i.e., the Akola slaughterhouse. These tissues
were collected irrespective of gross pathological lesions. Gross
pathological lesions were more common in bullocks slaughtered in
saline water area as compared to those slaughter in normal water
area. Histopathological examination of 120 liver tissues collected
from bullocks slaughtered at NWA and SWA showed: congestion
8.33 and 6.66%, haemorrhages 6.66 and 5.83%, granular degener-
ative changes 7.50 and 16.66%, necrosis 5.83 and 7.50%, abscesses
0.83 and 0%, cirrhosis 5.83 and 21.66% and hydatidosis 20.83 and
11.66%, respectively, thus indicating a higher percentage of his-
topathological alterations in liver tissues from bullocks collected
Page 483
 ESVCP Annual Meeting Abstracts

from the saline water area. Considering the increased number of ogy, Microbiology & Immunology, School of Veterinary Medicine,
sections showing perilobular connective tissue hyperplasia in the University of California, Davis, USA.
SWA group, a count of perilobular areas showing hyperplasia of
connective tissue and blood vessels was undertaken in high power Despite effective treatment for canine parvoviral enteritis, many
fields (1003). The average count per high power field in liver dogs still die of complications related to septicaemia or are
sections from the NWA and SWA groups was found to be 1.59 and euthanized because of anticipated high costs. More effective
2.29, respectively. The significantly increased counts of periportal prediction of the outcome of this disease will have an economic
and perilobular connective tissue proliferation in liver sections from impact if a prognosis can be determined early in the course of the
bullocks maintained in the saline water area indicated low-grade disease. A prospective study was performed on 62 puppies with
chronic hepatotoxicity through water salinity. The present in- confirmed canine parvoviral enteritis. Serial full blood counts were
vestigation thus indicated adverse effects of water salinity on the performed on a daily basis until death or discharge. Of the 11
health status of bullocks maintained in a saline water area, as puppies that died (18%), 9 died from complications of the disease
evident from histopathological changes of liver tissues. and 2 were euthanized due to financial restrictions and a poor
prognosis. Death occurred within the first 3 days of hospitalization
and a full post-mortem and histopathological examination were
performed on each case. Statistical analysis of the data showed that
in non-survivors (NS), the mean total leukocyte count did not rise
32 above 2.0 3 10 9/L. In the survivors (S), the total leukocyte count
FLOW CYTOMETRIC EVALUATION OF MORPHOLOGICAL FEA-
started rising within 24 hours of admission and often resulted in
TURES AND ANTIGENIC PATTERN IN CANINE LYMPHOMA.
a rebound leukocytosis. The NS remained lymphopaenic, whereas
M.E. Gelain1, M. Mazzili1, F. Riondato2, L. Marconato3,
the surviving puppies developed lymphocytosis within 24 hours of
S. Comazzi1. 1Dep. of Veterinary Pathology, Hygiene and Health,
admission, indicating an immune response. The NS also did not
University of Milan, Italy; 2Dep. of Animal Pathology, University of
develop monocytosis and remained severely eosinopaenic during
Turin, Italy; and 3‘‘L’Arca’’, Veterinary Clinic, Naples, Italy.
the course of the disease. Histopathology showed that most of the
NS had marked to severe thymic and lymphoid atrophy and
Cytomorphological features are widely used to identify and classify
marked to severe bone marrow hypocellularity. The red blood cell
lymphomas. However, criteria such as percentage of neoplastic
parameters were minimally affected in both groups. The study
cells, cell size and pleomorphism are difficult to standardize among
showed significant differences in the platelet count between S
different pathologists, quality of the smears and area of the slide
and NS. At 24 hours and 48 hours post-admission a significantly
examined. Flow cytometric analysis of fine needle aspiration (FNA)
higher percentage of NS showed marked thrombocytopaenia
samples could be a useful and minimally invasive technique to
compared with S. These results show that a reliable prognosis
provide more objective information together with cytological
could be obtained at 24 and 48 hours after admission by evaluating
examination. The aim of this study was to evaluate morphological
the total leukocyte, lymphocyte, monocyte, eosinophil and platelet
and immmunological features of different subtypes of lymphoma
counts.
by flow cytometry, comparing results to cytological examination.
Thirty-four neoplastic lymph nodal samples obtained by FNA were
submitted for flow cytometric and cytological analysis: 23 were B-
cell and 11 were T-cell neoplasms. According to the updated Kiel
classification, B-cell neoplasms were: 14 centroblastic, 1 centrocytic,
34
SEROPREVALENCE OF NEOSPORA CANINUM INFECTION IN
2 lymphoblastic, 2 macronucleolated medium-sized LSA and 2
DOMESTIC CAMELS (CAMELUS DROMEDARIUS) IN IRAN.
acute lymphoblastic leukaemia (ALL) infiltrating lymph nodes. T-
H.R. Haddadzadeh, Kh. Pirali, A. Sadrebazzaz, N. Sadreshir-
cell neoplasms were: 4 small clear cells, 5 pleomorphic, 1 large
azi, A. Gerami. Iran.
granular lymphocytes (LGL) lymphoma and 1 case of T-cell ALL
infiltrating the lymph nodes. Two lymphomas were of B-cell
One hundred serum samples were collected randomly from camels
lineage, but morphological diagnosis did not permit assignment
(Camelus dromedarius) in the biggest camel slaughterhouse of Iran,
of a precise entity. Forward scatter (FSC) properties were higher in
near Tehran (Varamin) from Sept. to March 2004. The camels had
centroblastic lymphomas compared with other entities. Lympho-
been transferred to the slaughterhouse from different parts of
mas defined as small cell lymphomas had a lower FSC, in particular
Iran: Khorassan, Hormozgan, Baloochestan and Booshehr. The sera
the small clear cell lymphomas. Analysis of immunopheotype
were used to evaluate IgG antibody against Neospora caninum using
revealed some unusual antigen expressions. In particular, dimin-
IFAT. Antibodies to N. caninum were found in 6% of camels: in 3
ished CD79a expression (10/21 B-cell lymphomas), CD34 expres-
camels (3%) at a titer of 1:20, in 2 (2%) at a titer of 1:40, and in 1 (1%)
sion by 4 B-cell lymphomas and other aberrant expressions, like
at a titer of 1:80 using whole N. caninum tachyzoites in IFAT slides
CD45dimCD3
CD4þCD8þ in a T-cell lymphoma, were noted.
(VMRD Inc., Pullman, WA 99163,USA). Of 100 studied camels, 83
Flow cytometry analysis on FNA samples could be useful not only
were male and 17 were female. Only 1 female was identified as
because it allows the contemporary identification of the antigenic
positive.
constellation and aberrant CD expression, but also because the more
precise definition of percentage and size of neoplastic cells is
minimally affected by smearing procedures.
35
COMPARISON OF MANUAL AND CELL-DYN 3500Ò RETICU-
LOCYTE ASSAYS IN HEALTHY DOGS AND CATS. D. Halmay,
33 T. Gaál. Department of Internal Medicine, Faculty of Veterinary
THE USE OF HAEMATOLOGICAL PARAMETERS AS PROG- Science, Szent István University, Budapest, Hungary.
NOSTIC INDICATORS IN CANINE PARVOVIRAL ENTERITIS. A.
Goddard1, A. Leisewitz2, N. Duncan3, M. Christopher4. Introduction: A reticulocyte (RET) count traditionally evaluated
Departments of 1Companion Animal Clinical Studies, 2Veterinary by manual method is an important index of erythropoesis and is an
Tropical Diseases, and 3Paraclinical Science, Faculty of Veterinary essential part of classification of anaemias. Background: The CELL-
Science, Onderstepoort, South Africa; and 4Department of Pathol- DYN 3500Ò haematology analyzer (CD) has a semi-automated

Page 484 Veterinary Clinical Pathology Vol. 35 / No. 4 / 2006

 ESVCP Annual Meeting Abstracts
RET-counting option for human patients based upon a supravital
staining technology. Objective: To evaluate the usefulness and
reliability of CD’s RET-assay in healthy dogs and cats and to compare
its results with those of the manual method. Materials: Twenty-six
healthy dogs and 16 cats were involved in the investigation. RBCs
and RETs were measured by the built-in programme of CD
(‘‘reticount mode’’) providing absolute the RET-count and RET-%.
For the manual method brillant-cresyl blue was used for staining
RETs on a blood smear. Three-hundred RBCs were counted on each
smear by two individuals and the RET-% was calculated. Results of
absolute (T/L) and relative (%) RET-counts for both methods were
compared. Results: The manual and CD absolute RET-counts in
dogs were 0.08 6 0.09 vs. 0.11 6 0.08 (NS) and in cats 0.17 6 0.19 vs.
0.09 6 0.04 (NS), respectively. Manual and CD RET-% in dogs were
1.15 6 1.5 vs. 1.58 6 1.26 (NS) and in cats 1.73 6 1.82 vs. 0.89 6 0.59
(p , 0.05), respectively. Discussion: Healthy dogs showed similar
RET-% measured by the two methods while in cats the manual
method expressed higher RET-% than CD. It is a default setting of CD
in reticount mode that RBC-count can be measured when it is less
than 10 310 12/L. Unfortunately, in cases of higher RBC-count RET-
% results can hardly be accepted. Conclusion: While absolute RET
values were similar in dogs and cats provided by the CD and manual
methods, we found that in feline blood due to the frequent presence
of macroPLTs or PLTaggregation the RBC-count from the CD is often
falsely increased; therefore, the RET-% is lower. Further studies on
a higher number of blood samples should be performed using both
methods in healthy and anaemic patients.
36
SEROPREVALENCE OF TOXOPLASMA GONDII INFECTION IN
OSTRICH FARMS AROUND TEHRAN. M. Ahmadi Hamedani,
H.R. Haddadzadeh, P. Khazraiinia. Departement of Clinical
Pathology, Faculty of Veterinary Medicine, University of Tehran,
Tehran, Iran.
Toxoplasma gondii is a coccidian parasite that infects a wide range of
warm-blooded animals including birds. Cats and other genera of
the family Felidae have been identified as definitive hosts of the
parasite. Humans can become infected through oocysts of the
parasite and/or consumption of raw and semi-cooked meat of
infected intermediate hosts. Ostriches like other birds can be an
intermediate host of the parasite. In the present study, 200 blood
samples were taken from ostriches from farms around Tehran and
tested for IgG antibody against T.gondii using IFAT. No positive case
was observed at a 1:20 titer. The eggs of ostriches have been
imported to Iran currently for the first time to establish their hus-
bandry. It is expected that Toxoplasma infection will be introduced
to ostrich farms of Iran in the future via oocyst-shedding cats.
37
HEMOGLOBIN TYPING IN IRANIAN STARLINGS. P. Khazraii-
nia, M. Ahmadi Hamedani, M. Haidarpour, M. Ahmadzadeh.
Department of Clinical Science, Faculty of Veterinary Medicine,
University of Tehran, Tehran, Iran.
Blood samples were taken from 16 starlings. Total hemoglobin was
measured and hemoglobin types were determined by the cellulose
acetate electrophoresis method using tris glycine buffer (pH58.6).
Two separate bands were observed, but unlike mammalian
hemoglobin types, the direction of their movement was toward
the cathode pole. The test was repeated but this time the samples
were located on anode pole. Two hemoglobin fractions moved
toward the cathode pole again. Probably the structure of avian
Vol. 35 / No. 4 / 2006 Veterinary Clinical Pathology
hemoglobin is different from mammalian hemoglobin and even
at pH 5 8.6 its charge is positive.
38
THE INFLUENCE OF CHICORY ON HEMATOLOGICAL PA-
RAMETERS IN BROILERS WITH HEPATOTOXICITY INDUCED
BY TETRACYCLINE. P. Khazraii nia, M. Heidarpour, M.
Ahmadi, S. Saei, A. Bahonar. Departement of Clinical Sciences,
Faculty of Veterinary Medicine, University of Tehran. Tehran, Iran.
This study was designed to examine the effects of chicory (Chicorium
intybus) powder on hematological parameters in broilers with
experimentally induced hepatotoxicity. Eighty one-day-old broilers
(approximately the same weight) were chosen. They were divided
randomly into 4 groups of 20 chickens each. All of them were fed
a normal diet for one week and on the second week, group one
chickens were fed a normal diet as the control group. The second,
third and fourth groups received tetracycline (4g/lit of water).
Blood samples were collected on 15th day. At the third week, group
one chickens were continued on the normal diet as the control group
and the second and third groups given the overdosage of
tetracycline had 2% and 1% chicory powder, respectively, added
to their diets. The fourth group was continued on the diet contain-
ing tetracycline only. Blood samples were collected on the 21st day.
PCV, RBC counts, WBC counts and WBC differential counts were
measured on the 5th and 21st day blood samples. There were no
significant differences in measured hematological parameters
between different groups.
39
TWO CASES OF FELINE ANAPLASMOSIS IN AUSTRIA: DI-
AGNOSIS AND EPIDEMIOLOGICAL ASPECTS. G. Kirtz1, P.
Ludwig1, M. Meli2, H. Lutz2, B. Czettel1, D. Thum1, E.
Leidinger1. 1Invitro Veterinary Laboratory, Vienna, Austria; and
2
Laboratory of the Veterinary Medical University, Zurich, Switzer-
land.
Anaplasma (Ehrlichia) phagocytophilum is a frequent infectious agent
in dogs in Austria, transmitted by Ixodes ricinus. We idenitifed
Anaplasma in 2 cats for the first time in Austria. Both animals were
from the southern part of the country and presented to the referring
veterinarian with fever, anorexia and a history of tick infestation.
Hematological findings were a marked thrombocytopenia and
a moderate regenerative anemia. In one case morulae could be
shown in neutrophils on a blood smear. Serology was done by
indirect immunofluorescence, and the infectious agent was identi-
fied by Taqman-PCR. Further data on the seroprevalence of
Anaplasma phagocytophilum in cats are being collected at the
moment. In areas with a high rate of anaplasmosis in dogs or
humans the disease should be considered as an important
differential diagnosis for other feline infectious diseases.
40
EOSINOPHIL COUNTS IN CATS BY MANUAL METHODS, THE
HEMAVET 950 AND LASERCYTE ANALYSERS. C. Lafond, J.
Coillard, P. Murgier, N. Bourgès-Abella, C. Trumel, J.P.
Braun. Département des Sciences Cliniques, Service de Médecine
des Carnivores Domestiques, Ecole Nationale Vétérinaire, Toulouse,
France.
Eosinophilia is an uncommon haematological change in cats but has
high medical significance in various diseases. Most blood analysers
have not been validated for eosinophil counting in animals. This
Page 485
 ESVCP Annual Meeting Abstracts
study was designed to compare two manual methods and two
analysers, the Hemavet and LaserCyte. Eosinophil counts were
performed on 62 EDTA-K3 feline blood samples by manual counts
on blood smears and manual counts after Phloxine B 1% staining,
and using the Hemavet 950 (impedance technology, Drew Scientific,
UK) and the LaserCyte (flow cytometry technology, Idexx Labora-
tories, USA) analysers. Results were compared by Passing Bablok’s
regression procedure and Bland Altman plot. The total WBC was
much higher with the Hemavet than with LaserCyte (paired t-test ,
0.001, mean difference of 3.9 3 10 9/L WBC). The agreement be-
tween the eosinophil manual count on the blood smear (x) and the
analysers (y) was poor for the Hemavet and good for the LaserCyte.
The Passing Bablok’s equations were [CI 95%]: y50.867 [0.518–
1.305] 3 þ0.009 [
0.202–0.117] for the Hemavet; and y50.981
[0.775–1.268] 3 þ0.220 [0.116–0.244] for the LaserCyte. Correlation
was better with the LaserCyte (r50.65) than with the Hemavet
(r50.32). There was no significant difference in eosinophil count
between Phloxine and Hemavet (paired t-test . 0.05), whereas it
was significally higher with the LaserCyte (paired t-test , 0.001).
The clinical classification of values as ‘‘normal’’ or ‘‘high’’ at the
1.5 3 10 9/L threshold showed discrepancies according to the
method but this probably results from the differences in total WBC
count. This study suggests flow cytometry gives a better estimate of
eosinophil counts in cats than impedance technology. However, it
must be investigated in larger numbers of eosinophilia cases.
41
A SURVEY OF FELINE HYPERTHYROIDISM IN AUSTRIA: 1995–
2005. E. Leidinger, J. Leidinger, P. Ludwig. Invitro Veterinary
Laboratory, Vienna, Austria.
Hyperthyroidism in cats is caused by thyroidal adenomas in older
cats (.8 years) and is the most common feline endocrinopathy. The
disease was only very sporadically diagnosed in Austria before
1995. Our survey, therefore, shows some interesting aspects of an
‘‘emerging’’ disease over a period of 10 years. We analysed data of
more than 100,000 cats submitted by veterinarians from all over
Austria to our laboratory. In about 1800 cases hyperthyroidism was
diagnosed (T4 concentration .50 nmol/l). Data on the prevalence
in comparison to other countries, breed, age, sex and additional
biochemical parameters that frequently are raised in hyperthyroid-
ism (ALP, ALT) are presented in this retrospective study. We also
show an unusual topographic distribution of hyperthyroidism in
Austria.
42
DETERMINATION OF ELECTROPHORETIC PATTERN AND
IMMUNODOMINANT ANTIGENS OF HAEMONCHUS CON-
TORTUS. B. Meshgi, S.H. Hosseini, A. Eslami. Department of
Parasitology, Faculty of Veterinary Medicine, University of Tehran,
Tehran, Iran.
Heamonchus contortus is a worm which may cause severe anemia
leading to the death of infected animals and economic losses. Its
diagnosis may be performed through parasitological and serolog-
ical tests as well as clinical symptoms. The objective of this study
was to determine the immunodominant and diagnostic antigen of
H. contortus. In the present study 4 different antigens including
the whole antigen of male and female as well as uterus and gut
were used. To collect positive serum, 10,000 third stage larvae were
administered to five lambs and two lambs free of infection were
used as the control group. SDS-PAGE and western blotting
were used for determination of electrophoretic patterns and specific
antigen of H. contortus. Our findings showed the presence of 4, 5, 11
Page 486 Veterinary Clinical Pathology
and 2 protein bands for whole antigen of male and female, uterus
and gut, respectively. In immunoblotting common bands were
observed between 45–90 KDa, and also there was a 35 KDa band for
gut antigen and a 110 KDa band for uterus antigen. This result
showed that the protein band with 35 KDa molecular weight can be
used as a diagnostic antigen in the serological studies of infection
with H. Contortus.
43
NEUROLOGICAL, CYTOHISTOLOGICAL, AND IMMUNOPHE-
NOTYPICAL CHARACTERIZATION OF SPINAL LYMPHOMA
IN A CAT. B. Miniscalco, F. Sammartano, A. Valazza, M.T.
Capucchio, E. Maggi, F. Riondato, R. Guglielmino. Dip.
Patologia Animale, Grugliasco, Torino, Italy.
A DSH male 8-year-old FeLVþ cat was referred for paraparesis and
urinary and fecal incontinence. Neurological findings localized the
lesion in the lumbosacral segments of the spinal cord (L4–S3).
Radiography of the thoracolumbar spine was normal, while
myelography revealed an abrupt and symmetrical interruption of
contrast at the level of the 2nd lumbar vertebra. No alterations of the
CSF were detected. The cat was euthanized. Anatomo-pathological
investigations revealed an extradural proliferation (EM) involving
the nerves of the cauda equina, the sacral spinal ganglia and
spreading cranially to L2; a mesenteric well defined gelatinous mass
(AM) and enlarged mesenteric lymph nodes (LN) were present.
Cytological examination of AM and EM revealed a monomorphous
population of medium to large-sized lymphocytes with basophilic
cytoplasm, irregularly clumped chromatin and prominent nucleoli.
Small lymphocytes and macrophages were also present in EM. LN
presented a reactive pattern. Histologically, EM and AM consisted
of round to polygonal cells classified as atypical immature
lymphocytic cells organized in sheets, separated by a thin fibrovas-
cular stroma, with disseminated necrotic areas and cellular debris.
More differentiated cells, plasma cells and macrophages were
frequently observed in EM. The proliferation involved the lepto-
meninges and, at the lumbar level, also the neuroparenchyma
showing a perivascular distribution. Both the cytological and
histological diagnosis was lymphoma for EM and AM, with
a reactive mononuclear population in EM, and reactive lymph
node for LN. Neoplastic cells were CD79aþ (IHC) and CD21þ (flow
cytometry); thus a diagnosis of B-cell LSA was made. Finally, FeLV
infection was confirmed by PCR on peripheral blood cells; proviral
sequences were detected by PCR also in EM, AM, and LN.
44
SERUM REACTION OF BOOPHILUS ANNULATUS-INFESTED
CATTLE TO PROTEINS OF DIFFERENT TISSUES. S. Nabian,
A. Nikpey. Department of Parasitology, Faculty of Veterinary
Medicine, University of Tehran, Research Center for Ticks and
Tick-Borne Diseases (RCTTD), Tehran, Iran.
The tick Boophilus annulatus is an important ectoparasite of cattle
that is present in West Africa, Central Africa, Asia and certain parts
of Southern Sudan. Many investigations have been done on
acquiring resistance to Boophilus micropolus in infested cattle. In this
study responses of infested cattle against soluble salivary gland,
soluble larval, and ovary extracts of Boophilus annulatus antigens
were tested. It has been shown that these ticks induce an immune
response in infested animals. Four cattle were each experimentally
infested with 10,000 Boophilus annulatus larvae and their humoral
immune response to salivary gland, ovary and larval extracts
during infestation were determined by an enzyme-linked immuno-
sorbent assay (ELISA) at different intervals. Salivary glands and
Vol. 35 / No. 4 / 2006
 ESVCP Annual Meeting Abstracts
ovary were dissected from ticks in cold PBS and rinsed in fresh PBS,
and then these organs and tick larvae were homogenized in PBS
containing 1 mM phenylmethylsulphonyl fluoride at 48C following
sonication for 10 min on ice with 40 W. The homogenates were
centrifuged at 10,000g for 30 min at 48C, and the supernatants were
stored at
708C. The protein content of the extracts was determined
using the method of Bradford (1976). Positive and negative
reference sera from infested and uninfested cattle were used to
standardize the enzyme immunoassay. The ELISA, using different
tissue preparations as antigens, showed high sensitivity. Our results
showed a positive reaction with sera from cattle infested with 8 ticks
as well. All extracts from salivary gland, larvae, and ovary showed
positive reactions with all sera tested starting from the first week
of infestation. Interestingly, in all cases a slight decrease in the
antibody level was observed within the second and third week of
infestation. After this time the level of antibody increased. These
results suggest that common antigens are present in different tissues
of B. annulatus.
45
USE OF C-REACTIVE PROTEIN (CRP) AND TOTAL SIALIC ACID
(TSA) TO DETECT MALIGNANCY IN CANINE EFFUSIONS.
M.D. Parra1, F. Tecles1, L. Soler2, K. Papasouliotis2,
C. Trumel3, J.J. Cerón1. 1Animal Medicine & Surgery De-
partment, Faculty of Veterinary Medicine, Murcia, Spain; 2De-
partment of Clinical Veterinary Sciences, University of Bristol, UK;
and 3Département des Sciences Cliniques des Animaux de Sport et
de Loisirs, Ecole Nationale Vétérinaire, Toulouse, France.
Introduction: In human medicine, CRP and TSA have been
reported to provide useful information for differentiating benign
and malignant effusions. In veterinary medicine canine CRP has
been evaluated in effusions showing differences among transudates,
modified transudates and exudates but has not been used to detect
malignancy, while TSA has not been investigated in effusions. The
aim of this study was to evaluate the diagnostic utility of CRP and
TSA in distinguishing effusions from dogs with malignancies and
non-malignant diseases. Materials: 61 canine pleural and abdom-
inal effusions (32 neoplastic and 29 non-neoplastic) were assessed.
CRP was measured by using a time-resolved immunofluorometric
assay (Parra et al, 2006), whereas TSA levels were determined by
using a colorimetric assay (Roche, Germany). Results: No signif-
icant differences were found in CRP (P 5 0.66) or TSA (P50.52)
values between neoplasic and non-neoplastic conditions. CRP
concentrations ranged between 0.051–56.85 lg/ml and between
0.055–48.37 lg/ml in neoplastic and non-neoplastic effusions,
respectively. TSA values varied between 58.26 and 4382.8 mg/L in
the tumoral group and between 57.16 and 4303.28 mg/L in the non-
tumoral one. Discussion: Our attempt at using CRP and TSA for
helping in the diagnostic work-up of effusions failed. In human
medicine studies supporting and detracting the usefulness of CRP
and TSA have been described but the cause of this discrepancy is not
fully understood. Further studies should be performed to assess any
possible correlation of canine CRP and TSA with the size or
invasiveness of the tumours. Conclusion: CRP and TSA measure-
ments in canine effusions are not suitable for the diagnosis of
malignancy.
46
EVALUATION OF WHITE BLOOD CELLS IN JUVENILE BARN
OWLS (TYTO ALBA). Z. Szabó1, A.  Klein2, A. Beregi1, T. Gaál1.
1
Department of Internal Medicine Faculty of Veterinary Science,
Szent István University, Budapest, Hungary; and 2Department of
Vol. 35 / No. 4 / 2006 Veterinary Clinical Pathology
Systematic Zoology and Ecology, Eötvös Loránd University, Buda-
pest, Hungary.
Introduction: WBCs in blood smears of barn owls (Tyto alba) have
not been studied so far. Objective: WBC parameters of 80 (38
female and 42 male) captive juvenile owls aged between 30–70 days
were determined for establishing reference intervals for juveniles of
this species. Materials: Blood samples were collected from the
wing veins of manually restrained birds into EDTA tubes using a 24-
G needle. Blood smears were fixed in methanol and stained with
Giemsa solution. The WBC count was estimated on the blood
smears by counting the number of WBCs per high power field (403)
in ten different fields. Differentials were evaluated by examining 300
leukocytes. Gender of the birds was determined by PCR amplifi-
cation of CHD-W and CHD-Z genes using either the P2/P8 or the
2550F/2178R primer pairs. Results: The estimated WBC count
was 11.5 6 5.4 3109/L. Dominant WBCs were heterophil
granulocytes (54.1 6 11.6%) while lymphocytes, monocytes and
eosinophil granulocytes were 41.8 6 11.3%, 3.6 6 3.3% and 0.5% 6
0.9%, respectively. In 30–50-day old birds (n525) the WBC count
was 13.7 6 5.9 3109/L while in the older ones (51–70-day-old,
n555) 10.6 6 4.9 3109/L (NS). The gender of the owls did not affect
the number and distribution of WBCs. Conclusion: Reference
intervals for WBC parameters of juvenile barn owls are established
and are similar to those of other owls.
47
EXPERIENCES WITH IMMUNOHISTOCHEMICAL EXAMINA-
TION OF P-GLYCOPROTEIN IN DOGS WITH HEMANGIOSAR-
COMA. D. Szécsényi, P. Vajdovich, T. Gaál. Department of
Internal Medicine, Faculty of Veterinary Science, Szent István
University, Budapest, Hungary.
Introduction: Immunohistochemical examinations were per-
formed to detect multidrug resistance protein 1 (MDR1), ATP-
binding cassette named P-glycoprotein (Pgp) on paraffin-embedded
splenic hemangiosarcoma samples obtained from splenectomized
dogs. Materials and Methods: We examined 8 samples obtained
from 6 different breeds (3 females, 5 males) by a double blind
method. Metastasis was found in five dogs. Mean age of the dogs
was 12 years. Immunohistochemical examinations were made by
using the avidin-biotin complex peroxidase method and a mouse
monoclonal antibody against Pgp (clone: C494). The degree of
expression of Pgp was indicated on a 4-point scale according to the
percentage of positively staining malignant cells. Score 1 was
assigned to 0–10%, 2 to 10–50%, 3 to 50–80%, and 4 to 80–100% Pgp
positivity. Results: Score 2 was found in 3 cases, score 2.5 in 2
cases, score 3 in 2 cases and score 4 in 1 case. The mean Pgp score
was 2.6. We found a positive correlation between the degree of Pgp-
expression and the presence of multiplex metastasis (r50.89, p ,
0.01). Discussion: We suggest that this method is suitable for
determining MDR in hemangiosarcoma cases to enable us to get
a closer prediction of the disease-free interval after chemotherapy
followed by splenectomy.
48
HEMOTROPHIC MYCOPLASMOSIS IN A FIV-POSITIVE CAT.
K. Thoren-Tolling. Clinical Pathology Laboratory, Bl Stjärnans
Animal Hospital, Gothenburg, Sweden.
A 12 year old cat, DSH, castrated male, was presented because of
poor appetite, signs of ataxia for 3–4 days, weakness, increased
water intake and weight loss over the last weeks. It was attacked
and bitten by another cat about 2 weeks earlier. The cat had
Page 487
 ESVCP Annual Meeting Abstracts
a normal vaccination record comprising FRV, FPL and FCV.
Laboratory data: RBC 3.6 3 10^12/L, hemoglobin 60 g/L, PCV
19% (MCV/MCHC 52/316 WNR), and moderate anisocytosis and
HJ bodies; leukopenia and neutropenia, WBC 1.8 3 10^9/L,
Neutrophils/segmented 0.8, bands 0.2, lymphocytes 0.9, and
monocytes 0.1 3 10^9/L; thrombocytes 72,000/lL; and reticulo-
cytes: 28,840/lL (RPI , 2). RBC morphology revealed Hemobarto-
nella organisms, and the cat was positive for Hemobartonella
haemominutum by PCR. The cat was FIV positive verified by
serology. Chemistry was normal except for albumin 24 g/L (Ref:
25–38) and phosphate 0.7 mmol/L (Ref: 0.85–2.60). Coagulation
times (APTT and PT) were prolonged. Red cell osmotic fragility
was markedly increased (Coombs’ test was not performed). Ultra-
sound revealed an enlarged spleen and liver with normal structure.
The cat was initially treated with vitamin K and i.v. fluids, and later
on with doxycycline. Discussion & Conclusion: The pronounced
anemia and the increased erythrocytic fragility were probably
related to the extracellular Hemobartonella organisms; the abnormal
chemistry data seemed related to the anorexia; and the leukopenia
was consistent with FIV infection. Hemobartonella organisms were
observed in moderate numbers and classified as Mycoplasma
haemominutum which is often seen with immunosuppressive
disorders and could under such conditions induce anemia and
haemolytic disease with clinical signs comparable to M. haemofelis,
normally considered more pathogenic. In Sweden, Mycoplasma
hemominutum is more often seen in cats than M. haemofelis. The
combination with FIV infection suggests the possibility of Hemo-
bartonella infestation of a primarily nonpathogenic, opportunistic
nature, developing into an acute haemolytic condition.
49
PROTOTHECOSIS IN A YOUNG DOG. K. Thoren-Tolling.
Clinical Pathology Laboratory, Bl Stjärnans Animal Hospital,
Gothenburg, Sweden.
Page 488 Veterinary Clinical Pathology
The dog, an Alaskan malamute, 1 year old intact female, was
presented because of weight loss and watery yellow diarrhea over
the last week, anorexia/poor appetite and occasional vomiting. The
dog had been roaming around freely about 10 days earlier.
Hematology was normal except for the PCV (57%). Chemistry data
(renal and liver panels) were normal, except ALP 4.6 lkat/L (Ref: 0–
4.2) and albumin 24 g/L (Ref: 25–38). TLI 46.5 lg/L (Ref: 5–45), B12
and Folate were within normal range. Amylase was 37.8 lkat/L
(Ref. 0–30 lkat/L). Rectal scraping and smears revealed an
inflammatory reaction with many squamous epithelial cells and
a mixed abnormal intestinal flora with round to oval extracellular
algal organism of various sizes, with a granular basophilic
cytoplasm and thin clear wall. Larger organisms may have
contained endospores. The size varied from 3–12 l wide and 3–15
l long, consistent with Prototheca organisms. Intestinal bacteria
included large numbers of short, fat, Gram-positive rods with
subterminal spores of Clostridium type, rods of Bacilli type and small
numbers of spiriliform bacteria. Additional findings included excess
fat droplets, fibrin threads and mucous material in addition to
inflammatory and epithelial cells. A fecal occult blood test revealed
small amounts of blood. The animal was treated with i.v. fluids,
trimethoprim/sulfa and diet prescription. The dog recovered and
regained appetite in about a week after treatment was initiated.
Discussion and Conclusion: The dog had probably eaten
contaminated material when roaming around in a muddy area
with water ponds and puddles. The alga Prototheca is ubiquitous in
the environment and may cause intermittent, often bloody diarrhea
in the dog. A disproportional number of cases have been reported in
Collies. The observed organisms were extracellular, but intracellular
location in macrophages and neutrophils is reported. Increased
populations of sporulated bacteria (Clostridium) mostly combined
with diarrhea/loose stools seems to be rather common in dogs in
Sweden and often combined with signs of malassimilation. The
prevalence of spiriliform bacteria is not known in this case and only
occasionally observed in our population of dogs.
Vol. 35 / No. 4 / 2006