Characterization of Concordant Visceral Leishmaniasis and Malaria Infections in Balb/C Mice

Abstract

Malaria and visceral leishmaniasis are the leading cause of mortality and morbidity associated with parasitic infections globally and where they coincide geographically, incidences of co-infection have been recorded. There is a paucity of data on the impact of these co-infections on public health. The objective of the present study was to evaluate clinical, parasitological outcome and humoral immune responses following Leishmania donovani (L. donovani) and Plasmodium berghei (P. berghei) co-infection in BALB/c mice. Mice were divided into three groups based on the experimental parasite: L. donovani- only infected mice; L. donovani- P. berghei mice (which were co-infected with P. berghei 60 days post-L. donovani infection) and P. berghei-only infected mice. A fourth (naïve) group was included to serve as a control. Following co-infection, 3 mice were sacrificed on days 0, 4 and daily from the 6th day till the end of the experimental period from each of the groups. Whole blood was used to prepare serum for Immunoglobulin gamma (IgG) quantification using Enzyme-Linked Immunosorbent Assay (ELISA) while tail prick blood sample were used for parasitemia determination in P. berghei groups. Spleen samples were obtained in all L. donovani groups for preparation of splenic impression smears for quantification of parasite load. Spleen, liver and brain tissues were obtained for histological analysis. Four mice from each group were left for studies on survival rates. Mean body weights were compared using a one-way analysis of variance (ANOVA) and adjusted with a Tukey post hoc test. Intergroup statistical analysis was tested using an independent t-test while a paired t-test was used to analyze intragroup means. For correlation analysis, Spearman’s rank correlation test was used while the Chi- square and Kaplan- Meier curve were utilized to compare the different survivorship rates. A P value ≤ 0.05 was considered statistically significant.Results revealed significant differences in body weights in both experimental and control groups (P< 0.0001). Independent t-test indicated significant differences in L. donovani parasite load (P<0.0021), P. berghei parasitemia (P= 0.0087) and P. berghei IgG responses (P= 0.0131) between experimental and control groups. However, results indicated no significant differences in mean L. donovani IgG responses in L. donovani- P. berghei co-infected mice. Intragroup data analysis showed significant differences in the body weight (P<0.0001), L. donovani parasite load (P< 0.0001), P. berghei parasitemia (P= 0.0418), L. donovani IgG (P< 0.0001) and P. berghei IgG responses (P= 0.0026) between the initial and termination dates in the L. donovani- P. berghei co-infected mice. There was a significant correlation between L. donovani parasite load and P. berghei parasitemia with their respective IgG responses (P=0.0258 and P=0.0245 respectively). Histological analysis revealed microhemorrhages, leukocyte sequestration, enlargement of red and white pulps, the formation of granulomas in the spleen and vascular degeneration in the liver which were less pronounced in the co-infected group. Co-infected mice survived longer as compared to P. berghei only but lesser than L. donovani- only mice group.Plasmodium berghei-only infected mice had the highest mortality rate compared to L. donovani- only or L. donovani- P. berghei infectedmice groups. Based on these findings, the study concludes that L. donovani and Plasmodium co-infection reduces disease severity and could possibly interfere with serological and parasitological disease diagnosis. Therefore, the study recommends that better disease control strategies and especially improvement of diagnostic procedures need to be expanded in regions of co-endemicity.