Bifidobacterium longum Administration Diminishes Parasitemia and Inflammation During Plasmodium berghei Infection in Mice

**Background:** During Plasmodium infection, infected erythrocytes sequester in gut tissues, leading to dysbiosis and altered intestinal immune responses. Probiotics such as Lactobacillus casei and Bifidobacterium longum have been shown to modulate gut immunity, but their effects during malaria infection are not fully understood. This study aimed to investigate the effects of L. casei and B. longum administration on parasitemia, gut microbiota composition, CD103 expression in intestinal dendritic cells, FoxP3 expression in T regulatory cells, and plasma IFN-γ and TNF-α levels in P. berghei-infected mice.

**Methods:** Six-week-old male C57BL/6 mice were acclimatized for 2 weeks and then randomly allocated into five groups (n=28 total). All groups except the negative control were infected intraperitoneally with 5×10^6 P. berghei ANKA cells. Treatment groups received either L. casei at 10^9 CFU/100 µL (group I), B. longum at 10^9 CFU/100 µL (group II), or a combination of both at 5×10^8 CFU/100 µL each (group III) orally from 5 days before to 6 days post-infection. Positive and negative control groups received PBS. Parasitemia was assessed daily via Giemsa-stained thin blood smears. At day 6 post-infection, mice were sacrificed, and intestinal tissues were collected. CD103 and FoxP3 expression in ileal samples were measured by direct immunofluorescence using PE-conjugated anti-CD103 and FITC-conjugated anti-FoxP3 antibodies. Plasma IFN-γ and TNF-α levels were determined by ELISA. Colon histopathology was assessed using H&E staining with a scoring system based on inflammation severity and architectural damage.

**Key Results:** All treatment groups showed increasing parasitemia from day 2 to day 6 post-infection. At day 2, parasitemia was significantly different among groups (p=0.001), with group II (B. longum) displaying the lowest degree of parasitemia. Significant differences in parasitemia persisted at day 3 (p=0.001), day 4 (p=0.001), day 5 (p=0.007), and day 6 (p=0.008). At day 6, group II showed the lowest parasitemia among all infected groups. Survival rates at day 6 differed significantly (p=0.04): positive control 72%, group I 67.2%, group II 67.2%, group III 100%, and negative control 39.52% (due to presumed cardiac stress). CD103 expression was significantly higher in all treatment groups compared to positive control (p=0.01), with group II showing the highest expression. FoxP3 expression also showed significant differences among groups (p=0.002), with group II and III showing increased expression compared to positive control and group I. Plasma IFN-γ and TNF-α levels were significantly lower in group II compared to positive control (p=0.022 and p=0.026, respectively), similar to negative control levels. Histological assessment showed that group II had significantly lower histological scores compared to positive control (p=0.05), with less erosion, ulceration, and inflammation. No significant differences in colon length were observed among groups (p=0.341).

**Clinical Implications:** This study demonstrates that B. longum administration in P. berghei-infected mice reduces parasitemia and modulates gut immunity by increasing CD103+ dendritic cells and FoxP3+ T regulatory cells while decreasing pro-inflammatory cytokines IFN-γ and TNF-α. These findings suggest that B. longum may have potential as an adjunctive therapy in malaria by supporting gut immune homeostasis and reducing inflammation. The study provides a basis for further research on probiotic supplementation in infectious disease immunity modulation. However, the study has limitations including the absence of a standard antimalarial drug control group, lack of measurement of T reg populations in the colon, and no analysis of gut microbiota composition or metabolites such as short-chain fatty acids. Future studies should investigate the colonization of these probiotics, their therapeutic effects compared to antimalarial drugs, and the mechanisms of immune modulation during Plasmodium infection.