Ethical statement
The current study was approved from the animal welfare and ethic society of University of Veterinary and Animal Sciences Lahore, Pakistan with No. DR 1112, Dated: 13-10-2017.
Selection of animals
A total of twelve piroplasm-free calves were undertaken in this study. The male crossbred calves of 4 to 6 months age were purchased from a local farm and were kept in experimental station of University of Veterinary and Animal Sciences Lahore, Pakistan. The animals were infected individually and collectively with ticks carrying
B. bigemina and
T. annulata reared at temperature 28 °C and at humidity >80% in Biological Oxygen Demand (BOD) incubator (Model ICO105 Memmert, Germany). The classification of the animals was done after screening of piroplasm infection in blood through PCR analysis. These calves were divided into four groups with each group carrying 3 animals. Group A consists of 3 crossbred splenectomized calf
(Kusminsky et al., 1995) infected with ticks (containing
B. bigemina) to use as a control positive, group B includes calves infected with ticks (containing
B. bigemina), group C consists of calves infected with ticks containing
B. bigemina and
T. annulata (mix infection), while group D consists of healthy animals free from any infection to use as control negative. All reared calves were in free access to water and corn silage.
Source of infection and parasitemia level in infected calves
Calves of group A, B and C were exposed to tick infestation. After 14 days pre-exposure with ticks, blood samples from experimental animals were collected in EDTA coated tubes. Thin blood smear of these samples were prepared to examine the PPE. To calculate the parasitemia level in infected calves and in cultured RBCs, three smears from each sample were prepared to count the average number of
B. bigemina/
T. annulata-infected RBCs according to previously used method
(Maharana et al., 2019). The PPE was measured by thin blood smear. Parasitemia level was calculated according to the previously reported formula
(Schetters et al., 1994).
Blood profile
Complete blood count (CBC) of samples from experimental animals was performed by using hematological analyzer (Abbott Cell Dyn 4000). The Hb concentration (gm/dl), PCV (%), RBCs count, WBC’s count and other erythrocytic indices (MCV, MCH, MCHC) were compared among the four experimental animal groups.
Catalase activity
Whole blood was collected from venipuncture of jugular vein of individual experimental animals (A, B, C and D groups) in EDTA-coated tubes. These samples were processed for identification of antioxidant status by measuring catalase activity by spectrophotometer at 374 nm absorbance. Linear differential equation was used to determine the catalase activity
(Hadwan and Abed, 2016).
Preparation of Medium 199, RPMI 1640 and DMEM
Medium 199 (Cat. No. 11150059), RPMI 1640 (Cat. No. 11875093) and DMEM (Cat. No. 11965092) were purchased from Thermo Fisher Scientific (Vanta, Finland). In this study, we cultivated the infected and non-infected RBCs in three different complete culture media following incubation in 5% CO2 at 37°C as previously conducted
(Adaszek and Winiarczyk, 2011; Erp et al., 1980; Vega et al., 1985). The complete culture media were prepared comprising of 60% (Medium 199, RPMI 1640 and DMEM) with supplementation of FBS 40%, amphotericin B 50 µg ml
-1, penicillin 100 IU, streptomycin 100 µg ml
-1 and HEPES 15 mM.
In vitro cultivation
Upon confirmation of infection and infection-free calves, blood samples were collected for
in vitro propagation of
B. bigemina in infected RBCs. A total of 10 ml whole blood was collected from the calves of all 4 groups in 15 ml EDTA coated tubes and thoroughly mixed. Live and dead cells were calculated by cell counting through hemocytometer
. For that, trypan blue was used as a marker for the differentiation of live and dead cells
(Rojas-Martinez et al., 2017). Centrifugation of collected blood samples was performed at 3000 rpm for 6 minutes at 4°C to obtain the pellet of cells. The cells were washed 3 times with 0.15 M phosphate buffer saline. The washed RBCs (80 µl) were then cultured within each type of complete culture medium (720 µl well-1) in a 24-well culture plate and incubated in 5% CO2 at 37°C. After every 24h, 700 µl of culture media was replaced with fresh complete culture media. The infected RBCs were sub-cultured
in vitro for 9 generations in duration of 36 days.
Osmotic fragility of infected RBCs
To perform erythrocytic osmotic fragility test, the most traditional protocol was followed
(Parpart et al., 1947). Briefly, NaCl solution at percentage of 0.9, 0.6, 0.5, 0.4, 0.3 and 0.0 in distilled water (dH2O) was used to optimize its concentration.
Lipid profile activity
The tests were performed for the profiling of three important parameters of lipids including cholesterol, triglycerides and high-density lipoprotein (HDL). Cholesterol analysis was done using Cholesterol LiquiColor® kit
(Quiclet et al., 2019). Triglycerides analysis was done by using Kit (ab65336)
(Zhang et al., 2015). HDL cholesterol level was measured by CHOLESTEROL liquicolor test kit (catalog no. INF 1001801 GB)
(Zhu et al., 2005).
Statistical analysis
RBCs of
B. bigemina,
T. annulata and in mix-infected calf were cultivated
in vitro in three different media to find out the propagation of parasitemia. Their results were expressed in the form of percentage with respect to their media. Graphical analysis was done by using the GraphPad Prism 7 software (La Jolla, CA 92037, USA). One-way ANOVA was performed to analyze the catalase activity, osmotic fragility and lipid profile of the infected RBCs. The significance among the groups was evaluated by using Post Hoc test (Tukey’s test). Significant values obtained were presented as *p<0.05, **p<0.01 and ***p<0.001 and “ns” represented non-significance (p>0.05).
