Bull Vet Inst Pulawy 55, 423-427, 2011
LIVER ENZYME ACTIVITY IN DOGS
INFECTED WITH BABESIA CANIS
WOJCIECH ZYGNER1, OLGA GÓJSKA-ZYGNER2, EWA DŁUGOSZ1,
1, 3
AND HALINA WĘDRYCHOWICZ
1
Division of Parasitology and Parasitic Diseases, Department of Preclinical Sciences,
Faculty of Veterinary Medicine,
Warsaw University of Life Sciences - SGGW, 02-786 Warsaw, Poland
[email protected]
2
Center of Small Animal Health Clinic Multiwet, 00-753 Warsaw, Poland
3
W. Stefański Institute of Parasitology, 00-818 Warsaw, Poland
Received: April 7, 2011
Accepted: July 21, 2011
Abstract
The influence of anaemia on alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase
(ALP) activities in dogs infected with B. canis was investigated. Samples of blood and serum from 230 infected dogs were divided
into two groups: A (with anaemia) and B (without anaemia). The differences in the activities of the enzymes between both groups
were not statistically significant. These results suggest that mild anaemia, as the only factor, has no influence on ALT, AST, or ALP
activity in canine babesiosis. However, this study certainly cannot exclude the possibility that a more severe anaemia can have a
major effect on the liver.
Key words: dogs, Babesia canis, babesiosis, hypoxia, aminotransferases, liver injury.
Babesia canis (formerly B. canis canis) is a
tick-transmitted parasite of dogs, causing a disease
called canine babesiosis. The disease also can be
caused by other species of the genus Babesia.
However, to date, only B. canis has been detected in
dogs and ticks in Poland (1, 2, 10, 26, 28).
The immunological response plays the main
role in the development of haemolytic anaemia during
the course of canine babesiosis (14, 23). Anaemia was
detected in 26.2%–31.4% of dogs infected with B.
canis in Poland (25). Elevated activities of alanine
aminotransferase (ALT), aspartate aminotransferase
(AST), and alkaline phosphatase (ALP) were detected
in 64.9%, 92.6%, and 31.7% of infected dogs,
respectively. These abnormalities result mainly from
liver damage (24, 29). Hepatopathy is a common
complication observed in canine babesiosis (14, 16),
and hypoxic injury is considered as the main reason of
hepatic disease (19). Anaemia observed in dogs with
babesiosis is considered as one of the factors causing
hypoxia and hypoxic liver injury, which can result in
an increase in ALT, AST, and ALP activities (19, 23).
In this study, the influence of anaemia on
increases in ALT, AST, and ALP activities in dogs
infected with B. canis was investigated.
Material and Methods
Two hundred and thirty samples of whole blood
and serum from 129 male and 101 female dogs,
infected with large Babesia, were collected. All
infected dogs were presented to the Center of Small
Animal Health Clinic Multiwet with clinical signs of
babesiosis including lethargy, anorexia or decreased
appetite, fever, pale mucous membranes, dehydration,
vomiting, and brown discoloured urine (due to
haemoglobinuria). The blood samples were collected
before treatment within 1–3 d of disease duration. The
first day when the dog was lethargic was considered as
the first day of the disease. Exclusion criteria were as
follows: any drug therapy in the preceding 4 weeks
(including dogs misdiagnosed with babesiosis), known
concurrent disease or infection, history of travelling
abroad in the preceding one year, and inability to
clearly identify anaemia (haematocrit within reference
interval and haemoglobin concentration below normal
values, and detection of dehydration in clinical
examination of non-anaemic dogs causing possible
masking of anaemia by dehydration). The inclusion
criteria were: diagnosis of babesiosis and collection of
blood samples for analysis before treatment.
EDTA was used as an anticoagulant. Serum was
obtained by the centrifugation of blood samples
424
without an anticoagulant. ALT, AST, and ALP
activities were determined by a clinical chemistry
analyser (XL 640, Erba Mannheim, Germany). The
diagnosis was based on the detection of Babesia
organisms in blood smears stained with Giemsa. The
level of parasitaemia was evaluated as it was described
in the previous study by Jacobson et al. (15).
Parasitaemia was expressed as the percentage of
parasitised red blood cells.
All blood samples were tested using PCR in
order to detect B. canis DNA. The DNA was extracted
from the blood using the Blood Mini kit (A&A
Biotechnology,
Poland)
according
to
the
manufacturer’s instructions. The efficiency of the DNA
isolation was confirmed by electrophoresis in a 1.5%
agarose gel. PCR was performed according to Sobczyk
et al. (21) with the primers BcW-A (5’-CAT CTA
AGG AAG GCA GCA GG-3’) and BcW-B (5’-TTA
ATG GAA ACG TCC TTG GC-3’) used to amplify the
18S rDNA gene fragment of B. canis. The expected
product was about 500 bp. As a positive control, the
DNA lysate from the blood of a dog infected with B.
canis was used. The infection in this dog was
confirmed by the sequencing of the PCR product,
which was found to be 100% identical to a fragment of
the B. canis 18S ribosomal DNA gene under accession
No. AY321119 in the GenBank® database. The size of
the PCR product was analysed by electrophoresis in a
1.5% agarose gel stained with ethidium bromide. The
PCR products with the expected amplicon size were
isolated from the agarose gel using the Gel-Out kit
(A&A Biotechnology). Next, 20 out of 230 PCR
products were randomly selected for sequencing in
order to verify the PCR test results. The sequencing
reaction was carried out on an AbiPrism® Genetic
Analyzer using the GeneScan® Analysis Software
computer programme. Obtained sequences were
compared to the sequence data available in the
GenBank®
using
the
BLAST
programme
(http://www.ncbi.nlm.nih.gov/BLAST/).
The red blood cell (RBC) count (normal values,
5.5–8.0 1012/L), haematocrit (Hct; normal values 0.37–
0.55 L/L), concentration of haemoglobin (Hb; normal
values 7.45–11.17 mmol/L), mean corpuscular
haemoglobin concentration (MCHC; normal values
19.8–22.3 mmol/L), and mean corpuscular volume
(MCV; normal values 60–77 fL) were calculated in
blood samples. These parameters were assessed with
an automatic haematologic analyser (Diatron®,
Abacus). Dogs with both haematocrit and
concentration of haemoglobin below reference values
(<0.37 L/L and <7.45 mmol/L, respectively) were
classified as anaemic.
The obtained results allowed dividing the blood
and serum samples into two groups: group A (anaemic
dogs), group B (non-anaemic dogs). The results were
analysed using the Statistica 8.0 programme. ShapiroWilk’s W test was used for the estimation of normality
in ALT, AST, and ALP activity distribution in groups
A and B. Wald-Wolfowitz Runs test was used to
compare the activity of the ALT, AST, and ALP in the
groups A and B, and to compare values of the
erythrocyte parameters in these groups. The value of
P<0.05 was considered significant. Results are depicted
as median and 25-75 percentiles.
Results
The dogs examined in this study belonged to
various breeds. The mean age of the dogs was 4 years
and 6 months (the youngest dog was 10 months of age
and the oldest was 11-year-old). Lethargy, anorexia, or
decreased appetite were present in all dogs. Ninety
(39.1%) dogs had anaemia (group A), and 140 (60.9%)
dogs had no anaemia (group B). A hundred and sixtysix (72.2%) dogs had fever (67 in group A and 99 in
group B). Haemoglobinuria indicating intravascular
haemolysis was detected in 16 (17.8%) dogs of group
A and in three (2.1%) dogs of group B.
The expected PCR product (500 bp) was
detected in all 230 blood samples. The 20 selected
product sequences showed 100% similarity to the 18S
rDNA partial sequence of B. canis isolated from dogs
in Poland.
The values of the erythrocyte parameters are
presented in Table 1. The mean level of parasitaemia in
groups A and B amounted to 0.13% ±0.04% and 0.12%
±0.04%, respectively. The differences in RBC count,
Hct and Hb concentration between groups A and B
were statistically significant. However, the differences
in MCV and MCHC between these groups were not
statistically significant.
Shapiro-Wilk’s W test showed that the
distribution of enzyme activity values was abnormal.
The activity of ALT within the reference values (3–50
IU/L) was found in 35 (39%) serum samples in group
A and in 42 (30%) samples in group B. An increased
ALT activity was detected in remaining serum samples
of both groups (61% and 70%, respectively). Two-fold
or greater increase in ALT activity was observed in 18
(20%) samples from group A and in 34 (24%) samples
from group B. The difference in the ALT activity
between both groups was not statistically significant
(P=0.0623; Table 2).
The activity of AST within the reference values
(1–37 IU/L) was present in seven (7.8%) serum
samples in group A and in eight (5.7%) samples in
group B. An increased AST activity was observed in
the rest of the samples. Two-fold or greater increase of
AST activity was detected in 50 (55%) samples in
group A and in 94 (67%) samples in group B. The
difference in the AST activity between both groups
was not statistically significant (P=0.526; Table 2).
The activity of ALP within the reference values
(20–155 IU/L) was detected in 61 (67.8%) serum
samples in group A and in 94 (67.1%) samples in
group B. An increased ALP activity was present in the
rest of the samples. Two-fold or greater increase of
ALP activity was detected in seven (97.8%) samples
from group A and in nine (6.4%) samples from group
B. The difference in the ALP activity between these
groups was not statistically significant (P=0.951; Table
2).
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Table 1
Statistical values of the erythrocyte parameters in groups A and B
Statistics
Group
RBC
Hct
Hb
MCV
MCHC
A
B
A
B
A
4.56
6.395
4.202
6.457
0.989
0.295
0.425
0.274
0.427
0.063
6.3
9.1
5.92
9.22
1.38
66
67
66.0
66.9
3.6
21.35
21.6
21.50
21.67
1.39
B
0.668
0.044
0.92
3.1
1.75
A vs. B
0.00*
5.5–8.0
0.00*
0.37–0.55
0.00*
7.45–11.17
0.8422
60–77
0.184
19.8–22.3
Mmed
µ
S.D.
p
Reference range
RBC – red blood cell count (1012/L), Hct – haematocrit (L/L), Hb – haemoglobin concentration (mmol/L), MCV – mean corpuscular
volume (fL), MCHC – mean corpuscular haemoglobin concentration (mmol/L), Mmed – median, µ - arithmetical mean, S.D. –
standard deviation, p – a value of P calculated for Wald-Wolfowitz Runs test, * - statistically significant differences, A – anaemic
dogs, B – non-anaemic dogs.
Table 2
Comparison of medians of ALT, AST, and ALP activities (IU/L) in groups A and B
Statistics
Mmed
25%-75%
Min.-Max.
p
Reference range
Group
ALT
AST
ALP
A
B
A
B
A
54.5
62.5
43 – 91
48 – 99
20 – 725
85.5
101.5
50 – 120
61.5 – 149.5
20 – 424
122.5
120
84 – 180
90 – 176
29 – 2295
B
20 – 502
25 – 498
37 – 763
A vs. B
0.0623
3 – 50
0.526
1 – 37
0.951
20 – 155
ALT –alanine aminotransferase (IU/L), AST –aspartate aminotransferase (IU/L), ALP –alkaline phosphatase (IU/L), Mmed – median,
25%-75% – 25th and 75th percentile, Min.-Max. – values of minimum and maximum, p – a value of P calculated for Wald-Wolfowitz
Runs test, A – group of anaemic dogs, B – group of non-anaemic dogs.
Discussion
The detection of B. canis DNA in all examined
samples is not surprising because B. canis is the only
piroplasm species detected so far in dogs in Poland (1,
26, 28). The occurrence of B. canis and the absence of
the other Babesia species infecting dogs result from the
fact that among the tick species transmitting these
pathogens, only Dermacentor reticulatus tick has been
detected in Poland (27, 30).
The prevalence of anaemia in the examined blood
samples is similar to the results of the previous studies
from Poland (11, 25, 31) but differs from the results
from Italy and Spain, where anaemia was detected in
74%–93.1% and in 11.1% of dogs infected with B.
canis, respectively (12, 20, 22). The difference in the
prevalence of anaemic dogs infected with B. canis
probably results from the different virulence of the
parasite strains and/or the duration of the infection.
The lack of significant differences in MCV and
MCHC values between groups A and B, and observed
means and medians of MCV and MCHC within
reference intervals in both groups, show that there was
no marked difference in the RBC count regarding the
relative number of immature erythrocytes in the blood.
This is shown by the lack of macrocytosis (increase in
MCV) and hypochromasia (decrease in MCHC), which
are typical for regenerative response after erythrocyte
destruction (6). The anaemia observed in this study was
normocytic and normochromic. This kind of anaemia is
typical at the start of haemolysis and appears to be nonregenerative (lack of immature erythrocytes) during the
first 2–3 d of haemolysis (6). In canine babesiosis
anaemia becomes regenerative (macrocytic and
hypochromic) after a few days of infection (23). Thus,
the values of MCV and MCHC observed in this study
within reference intervals indicate short duration (2–3 d)
of the disease. This was in agreement with the
information given by the owners of the dogs.
426
According to Allred (3) anaemia correlates with
parasitaemia in Babesia infected hosts. Moreover, Böhm
et al. (5) showed that high parasitaemia was
significantly associated with mortality among dogs
infected with B. rossi in South Africa. However, the
level of parasitaemia detected in this study, was not
different between groups A and B. This result is similar
to the results of the previous studies in Europe, which
demonstrated no correlation between parasitaemia and
severity of anaemia (9, 12). Jacobson (14) suggested that
anaemia and parasitaemia might correlate in
experimental infections.
The prevalence of elevated aminotransferase and
ALP activities in the examined serum samples is similar
to the results of previous studies from Poland and Italy
(12, 29). An increase in the enzyme activity above
reference values in the examined dogs suggests liver
injury. Both ALT and AST are present in high
concentrations in hepatocytes. Therefore, their leakage
into the circulation is observed when hepatocytes or
their membranes are damaged (4, 13). In this study, AST
activity increased in a larger number of serum samples
than ALT activity. This probably resulted from the fact
that AST is also detected in other tissues, such as
kidneys and RBC, which are also insulted during the
course of canine babesiosis (12, 23). However, if
haemolysis was a significant cause of increased AST
activity in serum of these dogs, there should be a higher
increase in AST activity in group A. Nevertheless, no
statistical difference in AST activity between both
groups was found. Moreover, the median of AST
activity was higher in group B. The observed higher
median of AST activity in group B may result from the
fact that this enzyme is also detected in the kidneys,
muscles, and pancreas (24). In group B, a 10-fold or
higher increase in AST activity was observed in seven
dogs, whereas, in group A only in one. It seems probable
that the kidneys, muscles, or pancreas could be also the
origin of AST in these dogs. Yet, the influence of
anaemia on these organs and tissues was not assessed in
this study. It is also probable that other undetected
diseases or opportunistic infections could additionally
influence the increase in AST activity. Thus, this result
shows that there is no influence of haemolytic anaemia,
or at least mild anaemia, on AST activity in dogs
infected with B. canis. ALT is a more specific marker of
hepatocellular injury in dogs (4, 8). Rajamohan et al.
(18) showed that ALT mRNA is also present in canine
heart, fat, brain, skeletal muscles, and kidneys.
However, in that study only ALT1 isoform expression
among two isoforms of this enzyme was investigated
and relative expression of ALT1 was noted as follows:
heart > liver > fat ~ brain ~ skeletal muscles > kidneys.
The authors of this publication suppose that the increase
in ALT activity in dogs infected with B. canis resulted
mainly from hepatocellular injury. Yet, anaemia had no
influence on liver damage.
ALP does not leak from the hepatocyte with
increased membrane permeability but the increase in this
enzyme activity is caused by the induction by drugs or
hormones. ALP derives from the bile ducts. Cholestasis
in canine babesiosis, probably caused by hepatomegaly,
could be one of the reasons of the marked increase in
ALP activity. This precedes the development of
hyperbilirubinaemia (4). The lack of a significant
difference in ALP activity between groups A and B
shows that anaemia had no influence on ALP activity.
The results of this study differ from the results of
previous investigations in which ALT, lactate
dehydrogenase, and ALP activities were compared in
anaemic and non-anaemic dogs infected with Babesia
rossi (19). The authors observed relatively higher liver
enzyme activity in anaemic dogs. However, means of
ALT and ALP activities were within reference values
both in anaemic and non-anaemic dogs. In this study, the
authors observed an increase in median ALT and AST
activities above reference values in both groups.
Medians of ALP activity were similar and within normal
values in both groups, and statistical analysis showed no
differences between these groups. The differences
between results of this study and results obtained by
Reyers et al. (19) probably result from the fact that
blood samples were taken from dogs infected with
different Babesia species. Moreover, Reyers et al. (19)
did not exclude the fact that destructed erythrocytes
might be the source of ALT and LDH in anaemic dogs.
The authors of this study investigated the
influence of anaemia on ALT, AST, and ALP activities.
Anaemia may be one of the reasons of hypoxia and
hypoxic liver injury observed during the course of
canine babesiosis. Yet, the results of this study did not
show correlations between anaemia and increased
aminotransferase and ALP activities. However, the
possibility that more severe anaemia can effect the liver
cannot be certainly excluded. Therefore, further
investigations dealing with hepatopathy in canine
babesiosis are necessary. Probably, TNFα, like in cases
of bovine babesiosis and human falciparum malaria,
may play the main role in liver damage causing
hypotension and oxidative damage (7, 14, 17).
Acknowledgments: The authors would like to
thank the staff of the Lab-Wet Centre of Veterinary
Diagnostics for their co-operation and assistance in the
collection of the dogs’ blood samples.
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