The study was designed as a single experimental, observational study which was conducted in the Department of Parasitology and Department of Pharmacology of the PGIMER, Chandigarh, India, for a duration of 1 year after taking approval from Institutional Animal Ethics Committee (IAEC), PGIMER, Chandigarh, vide Ref No. 81/IAEC/521. The total of 32 mice were obtained from Institutional Central Small Animal Facility, PGIMER, Chandigarh and were divided into four groups with 8 mice in each group. The mice were infected with 0.2 mL suspension of 106 parasitized erythrocytes intraperitoneally. Eight Balb/c mice were present in each of the four groups (i) GroupI (non-infected) (ii) GroupII (Infected group) (iii) GroupIII (P. berghei+ Lactobacillus casei) (iv) GroupIV (P. berghei+ Lactobacillus casei+ chloroquine). Parasitized RBCs were checked using the Giemsa staining technique under the light microscope. Laboratory standard cages were used for the housing of mice and acclimatized for 7 days. Standard livestock feed and clean drinking water were provided to them. This study was conducted according to Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines.
The mice were infected via injection with 0.2 mL suspension of 106 parasitized erythrocyte intraperitoneally. Samples of blood were taken from the tail of mice. Parasites were determined after preparation of thin blood smear and stained with Giemsa. PBS was administered to the mice in the first group (positive control) for four consecutive days. P. berghei infection was given to the second group. L.casei along with P. berghei was given to the third group. Chloroquine was administered to the mice at the dose of 15mg/kg once a day for four consecutive days of the fourth group . After completion of all the experimental procedures, animals were sacrificed by giving the anesthesia followed by cervical dislocation. This euthanasia procedure is done according to CPCSEA guidelines.
Determination of Parasitemia
Administration of extract was done after the collection of blood from the tail of each mouse which was inoculated with the parasite for about four days. For the determination of the baseline parasitemia, a thick and thin smear was made using the blood sample . By using the random field microscope, we have checked the parasitemia percentage by counting infected erythrocytes (parasitized) out of the 200 normal (non-parasitized) erythrocytes. The following formula used for the calculation of percentage parasitemia and average percentage parasitemia:-
For histopathological studies, the animals were sacrificed by cervical dislocation 3 days after the last dose on the fourth day of respective treatment and the organs were harvested. Pathological changes were observed in two organs i.e. liver, spleen. Tissue from each group was fixed in 10% formalin, embedded with paraffin. After routine processing paraffin sections from each tissue were cut into 5µm thickness and stained with hematoxylin and eosin. The photomicrographs of the relevant stained sections were taken with the aid of a light microscope . The following scores were used to grade the degree of histopathological changes or lesions observed in the organs: not observed (−), mild (+), moderate (++), and severe (+++).
Data analysis was done using statistical software SPPS Version 21. Representation of parasitemia count was done as mean± SD. Post hoc analysis by using ANOVA and Bonferroni multiple comparison test was used for the comparison of means. P<0.05 is considered statistically significant.