Canine babesiosis is a common and clinically significant tick-borne disease caused by haematozoan parasites of the genus
Our earlier study described the clinical course of babesiosis in dogs from areas of eastern Poland and selected aberrant haematological and biochemical parameters of the serum of dogs infected with this disease (4). The clinical picture of infection with
The aim of the study was to demonstrate a link between uncomplicated
The analyses of Zn and Cu trace elements levels were carried out using inductively coupled plasma-optical emission spectrometry (ICP-OES; iCAP 6000 series, Thermo Fisher Scientific, Waltham, MA, USA). Each measurement was performed three times and averages were used for the analysis. In the study, the appropriate wavelengths of the Zn and Cu elements (206.200 nm and 324.754 nm, respectively) were used for analysis in the ICP-OES device. Stock solutions for Zn and Cu trace elements were prepared from standard solutions (Chem-Lab NV, Zedelgem, Belgium) and distilled water was used as a blank solution. Calibration graphs were obtained from the ICP-OES device using blank and standard solutions and the concentration measurements of Zn and Cu trace elements were carried out according to the graphs. The blood plasma samples were diluted with distilled water. The standard solutions were measured again for system control after every five sample analyses. The concentration levels of Zn and Cu were expressed as μg/mL.
The activities of glutathione (GSH), SOD and catalase (CAT) were determined in whole blood in tubes with lithium heparin. Superoxide dismutase activity in the erythrocytes was determined using a commercial Ransod diagnostics kit (Randox Laboratories, Crumlin, UK), according to the manufacturer’s instructions, and CAT activity was determined according to Aebi (5). The plasma content of GSH was determined according to the methods described by Bartosz (6).
The real-time PCR reaction for all the isolated DNA samples was carried out using the Rotor-Gene RG6000 real-time DNA amplification system (Corbett Research, Mortlake, NSW, Australia) with SYBR Green 1 dye in thin-walled test-tubes with a capacity of 100 μL. A DyNAmo HS SYBR Green qPCR Kit (Finnzymes, Espoo, Finland ) was used to conduct the high-specificity reaction.
The reaction mixture, with a capacity of 20 μL, consisted of the following components: 2 μL of the DNA matrix, 7.2 μL of water, 0.4 μL of each of the GF2 and GR2 primers (final concentration of 50 pM), 10 μL of Master Mix containing a hot start version of the modified
The optimised real-time PCR reaction included 50 cycles, each comprising three stages: denaturation at 92°C for 60 s, annealing at 52°C for 60 s, and extension at 72°C for 90 s (3).
The PCR products were then purified using QIAquick spin columns (Qiagen), eluted in 50 μL of Tris 10 mM, pH 7.6, and sequenced at the Research Institute of the Polish Academy of Sciences, in Warsaw. DNA sequences were assembled and edited using SeqMan (Lasergene, DNAStar, Madison, WI, USA), and ClustalV alignments were generated to the published
All dogs in group 1 were apathetic. They also showed signs of anorexia, pyrexia, depression, and splenomegaly and had pale mucous membranes and dark red urine. The dogs in group 1 were injected subcutaneously with imidocarb dipropionate solution (Imizol; Schering Plough Animal Health, now MSD Animal Health, Kenilworth, NJ, USA), in a dosage of 5 mg/kg b.w., which resulted in recovery in all cases.
None of the dogs in group 2 showed any clinical signs of the disease. They were clinically healthy for the whole period of the study.
The results of haematological analysis showed some abnormally low levels in group 1 dogs but none in group 2 animals. A drop in haematocrit below 37% (the lower limit of normal) was found in every dog in group 1. A decrease of erythrocytes below 5.5 × 1012 (the lower limit of normal) was noted in 9 of the dogs with uncomplicated babesiosis. Leukopaenia (white blood cell count < 6 × 109) occurred in 6 sick dogs, while thrombocytopenia (platelet count < 200 × 109) was reported in all afflicted dogs (Table 1).
Results of haematological examinations of dogs from group 1 (dogs with babesiosis) and 2 (healthy dogs)
Group | Dog | WBC (×109) | RBC (×1012) | Ht (%) | PLT (×109) |
---|---|---|---|---|---|
1 | 6.8 | 6.76 | 38.7 | 43 | |
2 | 5.0 | 7.29 | 45.0 | 28 | |
3 | 6.9 | 6.20 | 38.4 | 24 | |
4 | 4.3 | 6.54 | 40.5 | 27 | |
5 | 6.2 | 5.39 | 32.0 | 30 | |
6 | 7.8 | 5.98 | 34.4 | 65 | |
7 | 10.3 | 6.40 | 39.2 | 35 | |
1 | 8 | 6.8 | 3.78 | 24.8 | 19 |
9 | 6.1 | 3.97 | 25.2 | 31 | |
10 | 9.3 | 4.30 | 25.6 | 39 | |
11 | 4.1 | 4.73 | 29.7 | 16 | |
12 | 7.7 | 5.08 | 32.4 | 48 | |
13 | 5.6 | 4.75 | 29.7 | 28 | |
14 | 4.8 | 5.07 | 31.7 | 29 | |
15 | 5.5 | 5.11 | 36.2 | 46 | |
16 | 10.4 | 6.40 | 42.1 | 231 | |
17 | 9.8 | 6.54 | 39.9 | 379 | |
18 | 8.9 | 6.86 | 44.4 | 224 | |
2 | 19 | 8.8 | 7.12 | 49.6 | 443 |
20 | 6.8 | 6.54 | 39.9 | 379 | |
21 | 9.9 | 6.16 | 42.1 | 220 | |
22 | 8.4 | 6.58 | 42.3 | 328 | |
Range | 6.0–17.0 | 5.50–8.50 | 37.0–55.0 | 200–500 |
WBC – white blood cells; RBC – red blood cells; Ht – haematocrit; PLT – platelets
Copper concentration was below normal in 14 out of the 15 dogs in group 1 (mean value 0.722 μg/mL) and was statistically significantly lower (P = 0.000247) than in the dogs in group 2, which all showed normal blood copper concentrations (mean value 1.392 μg/mL). The majority of the dogs in group 1 (13/15) had zinc blood concentrations below the lower limit of normal (mean value 0.478 μg/mL), whereas all the dogs in group 2 had normal values for this parameter (mean value 1.241 μg/mL). The differences were statistically significant (P = 0.000323) (Tables 2 and 3).
Cu and Zn concentrations in blood of dogs from groups 1 (dogs with babesiosis) and 2 (healthy dogs)
Dog | Cu 1.350 μg/mL | Zn 1.485 μg/mL | Group |
---|---|---|---|
1 | 0.788 | 0.400 | 1 |
2 | 0.713 | 0.463 | 1 |
3 | 0.578 | 0.249 | 1 |
4 | 0.650 | 0.379 | 1 |
5 | 0.818 | 0.455 | 1 |
6 | 0.658 | 0.463 | 1 |
7 | 0.524 | 0.404 | 1 |
8 | 0.823 | 0.905 | 1 |
9 | 0.724 | 0.610 | 1 |
10 | 0.662 | 0.466 | 1 |
11 | 0.684 | 0.383 | 1 |
12 | 0.790 | 0.380 | 1 |
13 | 0.812 | 0.622 | 1 |
14 | 0.792 | 0.410 | 1 |
15 | 0.816 | 0.587 | 1 |
16 | 0.894 | 1.412 | 2 |
17 | 1.120 | 1.184 | 2 |
18 | 1.830 | 0.923 | 2 |
19 | 0.993 | 1.340 | 2 |
20 | 1.751 | 0.882 | 2 |
21 | 1.344 | 1.612 | 2 |
22 | 1.812 | 1.332 | 2 |
CAT, SOD and GSH activity in dogs of groups 1 (dogs with babesiosis) and 2 (healthy dogs)
Dog | CAT (U/mL) | SOD (U/mL) | GSH (U/mL) | Group |
---|---|---|---|---|
1 | 9.6966 | 38.41 | 3.1 | 1 |
2 | 6.2483 | 38.70 | 1.5 | 1 |
3 | 7.9172 | 36.34 | 4.5 | 1 |
4 | 6.2483 | 33.52 | 3.7 | 1 |
5 | 2.6483 | 36.88 | 2.4 | 1 |
6 | 4.4690 | 38.16 | 3.4 | 1 |
7 | 2.4000 | 36.88 | 4.9 | 1 |
8 | 4.8828 | 34.98 | 3.6 | 1 |
9 | 3.8207 | 34.75 | 3.9 | 1 |
10 | 3.4414 | 34.25 | 5.9 | 1 |
11 | 4.2631 | 36.21 | 2.3 | 1 |
12 | 3.9343 | 38.18 | 3.1 | 1 |
13 | 4.9251 | 39.46 | 2.3 | 1 |
14 | 3.2887 | 39.14 | 2.1 | 1 |
15 | 6.1181 | 35.82 | 3.2 | 1 |
16 | 2.1241 | 34.57 | 3.0 | 2 |
17 | 0.8552 | 28.69 | 2.3 | 2 |
18 | 1.0000 | 31.51 | 3.6 | 2 |
19 | 0.6621 | 30.34 | 2.7 | 2 |
20 | 0.7448 | 31.72 | 2.2 | 2 |
21 | 1.8342 | 29.23 | 2.9 | 2 |
22 | 1.5261 | 31.51 | 4.4 | 2 |
CAT – catalase; SOD – superoxide dismutase; GSH – glutathione
Erythrocytic SOD activity in
The CAT activity in the erythrocytes of the dogs infected with
There was no significant difference (P = 0.458446) in the GSH activity between the infected and control dogs. The mean value of this parameter in the blood of dogs with babesiosis was 3.26 U/mL, whereas in that of healthy dogs it was 3.01 U/mL.
Based on the similarities between sequences of the 18S RNA gene fragment, five of the isolated
The aim of this study was to evaluate the possible changes in three antioxidant biomarkers (SOD, CAT, and GSH), as well as in Cu and Zn concentrations in dogs infected with babesiosis. The literature data on this subject in dogs are very scarce, and can be found in only two studies (8, 11). Dogs naturally infected with
Our own studies revealed significantly lower concentrations of Zn and Cu in the blood of dogs with babesiosis compared to the control group. In anaemic cattle infected with
Our studies demonstrated that in dogs with babesiosis, the SOD and CAT activity was higher than in healthy dogs. These findings conflict with the results presented by Crnogaj
Our observations are confirmed by the results of studies by Otsuka
We did not observe any correlation between the type of parasite strain causing the infection and the SOD, CAT, and GSH in the infected dogs, as happens with other clinical parameters (1, 4). Further studies in this respect are necessary, conducted on a larger group of animals.
In conclusion, the clinical study demonstrated that in dogs with uncomplicated early babesiosis, contrary to the biomarker picture in animals with advanced disease associated with MODS, the SOD and CAT activity were increased, while concentrations of Cu and Zn were reduced. Considering the strong negative correlation between the activity of antioxidants (SOD, CAT and GSH) in MODS cases and lethal outcome, it appears that the increased activity of SOD and CAT demonstrated in the study is a favourable prognostic, indicative of the uncomplicated form of the disease.