Sequential Leishmania major and Plasmodium berghei Co-infection Exacerbates Disease Severity, Parasite Burden and Haematological Alterations in BALB/c Mice

Abstract

Introduction: Malaria and leishmaniasis are important vector-borne parasitic infections in tropical and subtropical countries that often co-occur, yet their combined effects remain poorly understood. In the present study, we investigated the effects of sequential infection with L. major and P. berghei in BALB/c mice. Materials and Methods: We randomly assigned 160 BALB/c mice to four experimental groups: uninfected naïve controls (n = 40), Leishmania major mono-infected (n = 40), Plasmodium berghei mono-infected (n = 40), and co-infected (L. major and P. berghei) (n = 40). We infected mice with L. major in the right hind footpad; 21 days after Leishmania inoculation, once infection was established, we gave the co-infected and P. berghei mono-infected groups an intraperitoneal injection of P. berghei-parasitised erythrocytes. We monitored all animals daily, with assessments conducted over 13 days following malaria challenge. Primary endpoints included body weight change, Plasmodium parasitaemia, Leishmania parasite burden in footpad tissue, and comprehensive haematological profiling (red blood cell, platelet, and total and differential leucocyte counts). We compared groups using one-way or two-way ANOVA with post hoc comparisons as appropriate; p < 0.05 was considered statistically significant. Results: Co-infected mice developed more severe disease than either mono-infected group, with greater body weight loss: the mean body weight declined to 24.8 ± 2.4 g by Day 13, compared with 28.1 ± 1.5 g in L. major-mono-infected mice and 26.9 ± 1.9 g in P. berghei-mono-infected mice. Co-infected mice also had higher parasitaemia than P. berghei mono-infected mice (23.73 ± 3.02% versus 19.63 ± 3.39%; p = 0.008) and higher Leishmania parasite burden than L. major mono-infected mice (34.00 ± 1.38 versus 28.92 ± 6.16; p = 0.041). Co-infection induced anaemia, thrombocytopenia, leucocytosis, neutrophilia, lymphopenia, and monocytosis. Conclusions: Co-infection with L. major–P. berghei exacerbates disease severity, increases malaria parasite burden, and worsens haematological alterations, highlighting the need for an integrated approach to malaria–leishmaniasis co-infections in endemic areas.