Evaluation of Effect of Probiotics as an Add-on Therapy With Conventional Therapy and Alone in Malaria Induced Mice


 Objective: Chloroquine is used as conventional drug therapy for the treatment of malaria. The existence of resistance for chloroquine shown among various species of Plasmodium leads to the search for more efficacious therapy to treat malaria. Probiotics (Lactobacillus casei) have been tried as add-on therapy with chloroquine. Probiotics are ingested microorganisms associated with a beneficial effect on humans and other species. The study was done to check the efficacy of probiotics as an add-on therapy along with conventional drug therapy (chloroquine) to treat malaria.Results: Probiotics in combination with chloroquine showed complete suppression in parasitemia count. Representation of parasitemia count was done using mean ± SD. P<0.05 is considered significant. The results showed a reduction in parasitemia with probiotics treatment, which was further confirmed through histological observation of two major organs liver and spleen. Interestingly, further suppression of parasitemia and hemosiderosis was observed when probiotics were given along with chloroquine.


Introduction
Malaria is among one of the deadliest threats for the human species which is caused by various strains of malaria parasite e.g. P. falciparum, P. vivax, P. ovale, P. malariae, and P. knowlesi. The risk for developing malaria is about 3.3 billion all over the world [1]. The immune response plays a major role in the pathophysiology of malaria. Different immune protein which is released during the parasite attack can prohibit the growth of the same. Moreover, some signaling pathways act against the parasite e.g.
Tolls like receptor, signal transducers and activators of transcription pathway (STAT), Janus kinase pathway (JNK) [2]. Chloroquine was the choice of drug for the treatment of P. falciparum malaria but these days there is an emergence of resistance among P. falciparum species [3]. Microorganisms that are believed to provide health bene ts to the consumer are known as probiotics. Probiotics are generally gram-positive in nature and are isolated from gut micro ora known to provide an enhancement in the immune response to the consumer. Probiotics provide strain-speci c effectiveness as it is having immune stimulatory protection among pathogens of various types. It modulates intestinal microorganisms.
Probiotics have shown their effects on various epithelial cells, Payer's patches cells, and immune cells [4].
The result of this interaction is an increase in the number of IgA along with IgM [5]. Past studies had shown that gut microbiota correlates with the severity of malaria parasite infection. Probiotics have bene cial effects against malaria parasite infection [6]. Many studies which have speci c protocol towards understanding the molecular mechanism of probiotics needed to be done which involves clinical application also. The present study evaluates the effects of probiotics on parasitemia count, histopathological effects in malaria-infected mice and demonstrate the synergistic effect of probiotics along with chloroquine (conventional drug therapy) which can further lead to the development of xeddose probiotic combination for the treatment of malaria.

Study area
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.
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.

Treatment procedure
The mice were infected via injection with 0.2 mL suspension of 10 6 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 rst 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 [7]. After completion of all the experimental procedures, animals were sacri ced 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 [8]. By using the random eld 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:-Histopathology For histopathological studies, the animals were sacri ced 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 xed in 10% formalin, embedded with para n. After routine processing para n 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 [9]. 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
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 signi cant.

Results
Mainly 4 groups were taken for the study each containing 8 animals (Balb/c mice). (i) GroupI (noninfected) (ii) Group II (Infected group) (iii) Group III (P.berghei + Lactobacillus casei),(iv) Group IV (P.berghei + Lactobacillus casei + chloroquine). Parasitized RBCs were seen using light microscopy on the 4th day of inoculation by using the Giemsa staining technique on the microscopic slides. On the 1st day as compared to Group II (P.berghei treated), GroupIV (P.berghei + Lactobacilluscasei + chloroquine) has shown a statistically signi cant decrease (p < 0.01) in % parasitemia, On 2nd day, Group III ( P.berghei + Probiotic ) and Group IV (P.berghei + Lactobacillus casei + chloroquine) has also shown a statistically signi cant decrease in % parasitemia (p < 0.05 and p < 0.001 ) as compared to group II (P.berghei) while on day 3rd, Group IV has shown statistical signi cant (p < 0.01) decrease in % parasitemia as compare to group II. Finally, on the last 4th-day group IV has shown a statistically signi cant decrease in % parasitemia (p < 0.05) as compared to the P. berghei treated group as shown in Fig. 1. Overall, it has been shown that there is a reduction in parasitemia count when probiotics alone and probiotics along with chloroquine were given as compare infected group (P. berghei treated), shown in (Additional le 1: Figure  S1).
There were no changes found in the case of a control group as it has shown normal pathology (Fig. 2a &  3a). Hemosiderrhosis and Periportal in ammation in the liver section was found more in an infected group (2b) but there is a reduction of Hemosiderrhosis and Periportal in ammation when treatment of probiotic (2c) was given and further suppression was seen in the group treated with chloroquine and probiotics (2d). In the case of the spleen, megakaryocytic hyperplasia, lymphoid hypoplasia along Hemosiderrhosis have been seen in the infected group (3b). It has become mild and traces of Hemosiderrhosis pigments were seen when probiotic treatment was given (3c) but these were further reduced in the chloroquine and probiotics treated group (3d).

Discussion
This study is planned to show the synergistic effect of L. casei (Probiotics) along with chloroquine in malaria-induced mice. Probiotics were given 0.1ml for 3 days. A similar study was conducted by Oyetayo VO et al., [10] in which they showed Lactobacillus acidophilus and l.casei protective effects. Liver functions are improved by L.casei which is con rmed by Toxicological data of rat serum. The rat has been dosed with Lactobacillus were found to have a lowering of serum alanine aminotransferase activities which have value 15.50 and 18.27 IU/L as compare to that of the control. In the present study, probiotics treatment along with conventional drug therapy showed a statistically signi cant reduction in parasitemia count along with conventional drug therapy. There is an improvement in histopathological damages that are caused by the plasmodium parasite in organs like the liver and spleen. Hence, the present study con rmed that the use of probiotics as an add-on therapy along with conventional drug therapy has bene cial effects [10]. Probiotics treatment causes a reduction in the parasitic count and it also leads to suppression in parasitemia count. Chloroquine was given along with probiotics causes a further decrease in the parasite and thus complete suppression of the parasite takes place. Blood lm microscopic examination lead to the production of a mean parasitemia count which is found to be lesser than 2% within the period of 4 days before starting treatment while chloroquine + Probiotics (L. casei) cleared parasitemia on the third day of treatment [11]. A previous study done by Khalifa EA, 2016, has shown that as compared to the non-treated group, the L. casei treated group has decreased the parasite load in the infected mice. So, probiotics can be considered as a promising and hopeful alternative for the treatment of various parasitic diseases. Different standard drug therapies in combination with probiotics can provide a de nite treatment to eradicate the various parasitic infections [12]. Moreover, probiotics treatment has also been effective in treating bacterial infections as well e.g. Salmonella. Probiotics have several mechanisms of action for example, by increasing the production of acid which kills the acidsensitive bacteria's or by releasing the bacteriocins [13]. Another study which has been conducted in mice shown a reduction in the number of worms (about 33%) and egg output speci cally which was infected by S. venezuelensis by probiotics activity by improving the immune response but the factors responsible for these effects are still not clear [11].On the other hand, the study which was done by Juliette Guitard et al., has shown that administration of L.casei mixture (daily) was unable to eradicate the complete parasite load (Cryptosporidium parvum) in the neonatal rat model. The reason for this may be due to lack of production of INF-γ which could show the protective effect [14].
This study has shown that when L. casei is given along with the standard drug therapy (chloroquine) showed a synergistic effect in the mice model of malaria as it has reduced the parasitemia count and improved the pathological changes that appeared after getting the infection.

Limitations
The study includes the only preliminary nding that shows the only effect of probiotics on malaria parasite in vivo environment. However, lacks to depict host response while taking probiotics in case of malaria infection. A more elaborated protocol is required for further deeper investigations such as studying of involvement of innate and adaptive immunity through estimation of antibodies, T subset regulation, and cytokines estimation. Additionally, experiments such as a survival plot would better explain the usefulness of these prophylactic measures. Graph showing percentage Parasitemia. The data are represented as mean ± SD. Statistical Signi cance of data are given as *p < 0.05; **p < 0.01;***p < 0.001

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. ARRIVEGuidelinesChecklist.docx