INFLUENCE OF ANAEMIA ON AZOTAEMIA IN DOGS INFECTED WITH BABESIA CANIS IN POLAND

Bull Vet Inst Pulawy 53, 663-668, 2009 INFLUENCE OF ANAEMIA ON AZOTAEMIA IN DOGS INFECTED WITH BABESIA CANIS IN POLAND WOJCIECH ZYGNER AND HALINA WĘDRYCHOWICZ 1, 2 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 wojciechzygner@yahoo.pl 2 W. Stefański Institute of Parasitology, 00-818 Warsaw, Poland Received for publication May 5, 2009 Abstract Samples of blood and serum from 127 dogs infected with Babesia canis were divided into two groups: A (without anaemia) and B (with anaemia). Additionally, Group B was divided into two subgroups: B1 (with increased MCHC) and B2 (without increased MCHC). Erythrocyte parameters, such as red blood cell count, haematocrit, of haemoglobin, and mean corpuscular haemoglobin, were estimated by using an automatic haematologic analyser. and serum urea were determined in samples of blood serum with a clinical chemistry analyser. Student s t-test was used to compare the of creatinine and serum urea in groups A and B, and subgroups B1 and B2. The differences in mean serum urea and creatinine between these groups and subgroups were not statistically significant. The results of this study did not show any correlations between anaemia and azotaemia, or between increased MCHC and azotaemia. These results suggest that anaemia, as the only factor, has no influence on azotaemia in canine babesiosis. Key words: dog, Babesia canis, azotaemia, anaemia. Babesia canis (formerly B. canis canis) is a haemoprotozoan apicomplexan parasite, which infects dogs, causing a disease, named canine babesiosis. The disease can be also caused by other species of the genus Babesia, such as: B. vogeli (formerly B. canis vogeli), B. rossi (formerly B. canis rossi), B. gibsoni, and B. conradae (10, 20). Among all Babesia species, three occur in Europe: B. canis, transmitted by the tick Dermacentor reticulatus, B. vogeli, and B. gibsoni, transmitted by Rhipicephalus sanguineus (7). Only B. canis has been detected in dogs and ticks in Poland (2, 4, 17, 21, 23). Anaemia was detected in 26.2% 31.4% of dogs during the course of canine babesiosis in Poland (22, 24), and renal insufficiency, manifesting itself with increased of serum urea and creatinine, in 30.7% 62.4%. The immunological response plays a main role in the development of anaemia. Babesia initiates a mechanism of antibody-mediated cytotoxic destruction of circulating erythrocytes. Auto-antibodies are directed against components of the membranes of the infected and uninfected erythrocytes. This causes intravascular and extravascular haemolysis, which leads to anaemia, and intravascular haemolysis also leads to haemoglobinaemia (7, 14). Antibody-coated erythrocytes are destroyed by macrophages in the spleen and liver (extravascular haemolysis). Intravascular haemolysis results from the complementary binding to the erythrocytic membrane by the surface antigen antibody reaction (5). Renal insufficiency is observed in dogs infected with B. canis (7). Mathe et al. (13) detected acute renal failure in 31% of affected dogs. This nephropathy results mainly from the degenerative and necrotic changes of epithelial cells in the proximal renal tubules (12). Some authors also observed haemoglobinuric nephrosis. However, this change was observed in a minority of dogs (8, 12). In Poland, Abramowicz (1) detected an increased of serum haemoglobin in affected dogs, but did not find a correlation between intravascular haemolysis and nephropathy. Free serum haemoglobin can probably lead to haemoglobinuric nephrosis, but renal insufficiency also requires additional processes such as hypoxia and reduced renal perfusion to become clinically evident (1, 7, 12). In this study, the authors have investigated the influence of anaemia, as one of factors causing hypoxia, a renal insufficiency manifested in increased of serum urea and creatinine in dogs infected with B. canis. Material and Methods The authors collected 127 samples of blood and serum from 59 male and 68 female dogs from Warsaw infected with large Babesia. The dogs belonged to

664 various breeds, mainly such breeds as Labrador, German Shepherd, Beagle, Dachshund, Dalmatian, and crossbreed dogs. The mean age of the dogs was 5 years. The diagnosis was based on the detection of Babesia organisms in blood smears stained with Giemsa. Among the samples of blood, 13 were randomly selected for PCR testing in order to detect B. canis DNA. DNA was extracted from the blood using the Blood Mini kit (A&A Biotechnology) 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. (17) 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 revealed 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, five out of 13 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 Analyser using the GeneScan Analysis Software computer programme. The obtained sequences were compared to the sequence data available in the GenBank using the BLAST program (http://www.ncbi.nlm.nih.gov/blast/). The red blood cell (RBC) count, haematocrit (Hct), of haemoglobin (Hgb), and mean corpuscular haemoglobin (MCHC), were estimated in the samples of blood. These parameters were assessed with an automatic haematologic analyser (Diatron, Abacus). Ethylenediamine tetra-acetic acid (EDTA) was used as an anticoagulant. Samples of the serum were obtained by the centrifugation of blood samples without an anticoagulant. Serum creatinine and urea were determined with a clinical chemistry analyser (Pointe 2000, Pointe Scientific). The results obtained allowed us to divide the blood and serum samples into two groups: group A the blood and serum samples of dogs without anaemia, group B the blood and serum samples of dogs with anaemia. Additionally, group B was divided into two subgroups: subgroup B1 blood and serum samples of dogs with an increased MCHC above the reference value (observed in intravascular haemolysis), and subgroup B2 - the blood and serum samples of dogs with a MCHC within the reference value. The results were analysed using the Statistica 6.0 program and Microsoft Office Excel 2007. Student s t-test was used to compare the of the creatinine and urea in the groups A and B, and subgroups B1 and B2. The A P value of <0.05 was considered significant. Results The expected PCR product (500 bp) was detected in all 13 randomly-selected blood samples. The five selected product sequences showed 100% similarity to the 18S rdna partial sequence of B. canis isolated from dogs in Poland (accession No. AY321119). The values of the erythrocyte parameters are presented in Table 1. Forty-five out of the 127 dogs (35.4%) had anaemia (group B). Twelve out of the 45 dogs with anaemia (26.7%) had an increased MCHC (subgroup B1), and the rest of them (73.3%) had MCHC within the reference interval (subgroup B2). The values of the erythrocyte parameters in subgroups B1 and B2 are presented in Table 2. The mean RBC count (T/L) of all the blood samples (n=127) amounted to 5.46 ±1.47. The mean Hgb (mmol/l) in the 127 blood samples amounted to 7.82 ±2.06. The mean Htc (L/L) in the 127 blood samples amounted to 0.364 ±0.1). The mean MCHC values (mmol/l) in all of the blood samples amounted to 21.48 (±1.16). A creatinine within the reference values was present in 81 out of the 127 samples of serum (63.8%), and an increased creatinine was detected in 46 out of the 127 samples (Fig. 1). The mean creatinine s in groups A and B are presented in Table 3. The difference in the mean creatinine between these groups was not statistically significant. The mean creatinine s in subgroups B1 and B2 are presented in Table 4. The difference in the mean creatinine between these subgroups was not statistically significant. A serum urea within the reference values was present in 16 (12.6%) out of the 127 samples and an increased level of serum urea was detected in 111 out of the 127 (Fig. 2). The mean serum urea s in groups A and B are presented in Table 3. The difference in the mean serum urea between these groups was not statistically significant. The mean serum urea s in subgroups B1 and B2 are presented in Table 4. The difference in the mean serum urea between these subgroups was not statistically significant. The relationship and the trend line between haematocrit and creatinine in all the 127 blood samples are presented in Fig. 3. The correlation between the level of creatinine and haematocrit (R 2 ) amounted to 0.0035.

665 Table 1 The values of the erythrocyte parameters in groups A and B Parameter RBC (T/L) Hgb (mmol/l) Hct (L/L) MCHC (mmol/l) Group A B A B A B A B µ 6.192 3.924 8.88 5.63 0.415 0.259 21.37 21.75 SD 0.669 1.069 0.95 1.47 0.045 0.068 0.66 1.70 M med 6.08 4.37 8.75 6.2 0.41 0.29 21.4 21.3 Min. 4.98 1.08 7.4 2.3 0.35 0.11 19.3 19.3 Max. 7.65 4.92 11.4 7.3 0.54 0.34 22.7 27.6 µ arithmetical mean; SD standard deviation; M med median Min., minimum; Max., maximum Table 2 The values of the erythrocyte parameters in subgroups B1 and B2 Parameter RBC (T/L) Hgb (mmol/l) Hct (L/L) MCHC (mmol/l) Group B1 B2 B1 B2 B1 B2 B1 B2 µ 3.587 4.047 5.68 5.61 0.238 0.267 24.01 20.94 SD 1.338 0.947 1.612 1.437 0.072 0.066 1.42 0.83 M med 4.19 4.5 6.4 6.1 0.265 0.29 23.65 21.1 Min. 1.08 1.67 2.8 2.3 0.12 0.11 22.6 19.3 Max. 4.92 4.9 7.3 7.2 0.31 0.34 27.6 22.5 µ arithmetical mean; SD standard deviation; M med median; Min. minimum; Max. maximum Table 3 Comparison of mean creatinine s (mg/dl) and serum urea (mg/dl) in groups A and B Parameter Group A Group B Group A Group B µ 1.98 1.83 98.1 88.2 SD 1.61 1.51 91.7 89.3 M med 1.3 1.4 56.6 50.4 Min. 0.8 0.6 19.0 20.4 Max. 9.0 8.8 432.5 399 t 0.521 0.587 µ arithmetical mean; S.D. standard deviation; M med median; Min. minimum; Max. maximum; t value of t. A lack of differences between variances. Significance at P<0.05 Table 4 Comparison of mean creatinine s (mg/dl) and serum urea (mg/dl) in subgroups B1 and B2 Parameter Subgroup B1 Subgroup B2 Subgroup B1 Subgroup B2 1.98 1.77 121.5 76.0 SD 1.47 1.54 130.2 67.6 M med 1.25 1.4 46.4 53.0 Min. 0.8 0.6 20.4 21.0 Max. 5.2 8.8 399.0 280.0 t *0.410 **1.1551 µ arithmetical mean; S.D. standard deviation; M med median; Min. minimum; Max. maximum; t value of t. *A lack of differences between variances. ** A presence of differences between variance, a value of t 0.05 = 2.1868. Significance at at P<0.05

666 60 50 Number of serum samples 40 30 20 10 0-1 0 1 2 3 4 5 6 7 8 9 10 Fig. 1. The ceatinine in the 127 samples of serum (mg/dl). M med = 1.3; = 1.93; SD = 1.57; Min. = 0.6; Max. = 9.0; reference interval 0.5 1.7 (M med - median;, - arithmetical mean; SD - standard deviation; Min. - minimum; Max. - maximum). within the reference interval detected in 81 serum samples. 70 60 50 Number of serum samples 40 30 20 10 0 0 50 100 150 200 250 300 350 400 450 Serum urea Fig. 2. Serum urea (mg/dl). M med = 54.3; = 94.6; S.D. = 90.6; Min. = 19; Max. = 432.5; reference interval 8.0 28.0 (M med - median; arithmetical mean; SD - standard deviation; Min. - minimum; Max. - maximum). Serum urea within the reference interval detected in 16 serum samples.

667 Fig. 3. The relationship between haematocrit and creatinine in the blood and serum samples from all the 127 dogs (the line - trend line; diamonds - cases). Discussion The detection of B. canis DNA in all the examined blood samples is not surprising, because only B. canis DNA has been detected in Poland in previous studies (2, 4, 17, 21, 23). The occurrence of B. canis and the absence of the other Babesia species infecting dogs results from the fact that among the tick species transmitting these pathogens, only the D. reticulatus tick has been detected in Poland (19, 25). The prevalence of anaemia in the examined blood samples is similar to the result of the previous study from Poland (22) but differs from the results from Italy and Spain, where anaemia was detected in 74% 93.1% and in 11.1% respectively of dogs infected with B. canis (6, 15, 18). The mean values of the erythrocyte parameters are similar to the result of the previous studies from Poland and Spain (1, 15, 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 increase in the creatinine and serum urea above the reference values in the examined dogs shows azotaemia. There have probably been concurrent prerenal and renal azotaemias during the course of canine babesiosis. Dehydration, methaemoglobinaemia, and hypoxia observed in canine babesiosis are considered to be causes of renal dysfunction, which results in an increase in serum urea and creatinine (3, 6, 11, 12). The authors of this study investigated the influence of anaemia on azotaemia. Detected in anaemic dogs (group B), a low mean Hct, RBC count, and Hgb seem to be one of the causes of hypoxia. The results of this study did not show any correlations between anaemia and azotaemia. Moreover, there were no correlations between an MCHC, which had increased (above-reference) values and the serum urea or creatinine. These results suggest that anaemia, as the only factor, has no influence on azotaemia. The same observation concerns haemoglobinaemia, which is present in intravascular haemolysis (connected with an increased MCHC). The low correlation between the level of creatinine and haematocrit confirms these results. This correlation can result from dehydration. Schetters et al. (16) suspect that the correlation between the creatinine and haematoctrit results from the dilution of the blood as a consequence of hypotension. However, a decrease or increase in haematocrit is the result of dehydration and haemolysis, so it also seems possible that the azotaemia observed is correlated with dehydration. In the authors opinion dehydration can be one of the factors, but not the only one, influencing azotaemia in dogs infected with B. canis. The detection of azotaemia in this study probably mainly results from hypoxia. Anaemia and haemoglobinaemia seem to be not enough on their own to cause the development of renal insufficiency. A possible cause of hypoxia is hypotension observed during the course of canine babesiosis (9). Hypotension also causes dilution of the blood and the correlation between the level of creatinine and haematocrit (16).

668 The results of this study confirm the results of previous studies. Mathe et al. (12) observed the same histological changes in the kidneys of anaemic and nonanaemic dogs infected with B. canis. Schetters et al. (16) detected a correlation between haematocrit and the creatinine. These findings and the results of this study suggest that the development of renal insufficiency in canine babesiosis is not directly dependent on anaemia. Azotaemia observed in dogs infected with B. canis seems to be a consequence of decreased renal perfusion, which leads to renal hypoxia. Acknowledgements: The authors would like to thank the staff at the Lab-wet Centre of Veterinary Diagnostics for their co-operation and assistance in the collection of the dogs blood samples. References 1. Abramowicz B.: Contribution of haemolysis to kidney and liver disturbances in dogs with babesiosis. Medycyna Wet 2008, 64, 213 218. 2. 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