Abstract

Malaria and visceral leishmaniasis coexist in the same geographical regions. However, dual co-infection with parasites causing these diseases and their impact on public health is poorly documented. Interactions between these parasites may play a role in disease outcome. The present study set out to evaluate the clinical and immunological parameters following Leishmania donovani and Plasmodium berghei co-infection in BALB/c mice. Mice were divided into four groups; L. donovani- only, L. donovani-P. berghei, P. berghei- only and naïve. Body weight, parasite burden, total IgG, IFN-γ and IL-4 responses were determined. To determine the survival rate, four mice were used from each group. Tissues for histological analysis were taken from spleen, liver and brain. Results indicated significant differences in body weight (P<0.0001), L. donovani parasite load (P< 0.0001), L. donovani IgG (P< 0.0001), P. berghei parasitemia (P= 0.0222), P. berghei IgG (P= 0.002), IFN-γ (P<0.0001) and IL-4 (P<0.0001) in dual-infected mice. There was no correlation between L. donovani parasite load and IgG responses in single or dual infections, while there was a positive relationship of P. berghei parasitemia and IgG responses in the dual infection group only. Plasmodium berghei had the highest mortality rate compared to L. donovani- only and L. donovani- P. berghei infected mice groups. Histological analyses showed enlarged red and white pulps and pathological changes in the spleen, liver and brain tissues which were less pronounced in co-infected group. We conclude that L. donovani and P. berghei co-infection reduces disease severity and these changes seem to correlate with variation in serum IgG and cytokines (IFN-γ and IL-4). Therefore, the study recommends the importance of inclusion of early screening of malaria in Visceral Leishmaniasis patients in regions where malaria is co- endemic.

1. Introduction

Visceral leishmaniasis (VL) commonly known as kala-azar is a systemic disease transmitted by phlebotomine female sandflies ¹. It is affecting millions of people globally and comes second after malaria in terms of casualties caused by parasitic diseases ². It is caused by Leishmania infantum and L. donovani in the old world and the latter is almost always fatal if left untreated ³. East Africa (Kenya, Ethiopia, Somalia, Sudan and South Sudan), South-East Asia Region (Pakistan, Bangladesh and India), South America (Brazil, Colombia and Peru) and Mediterranean region (Saudi Arabia, Afghanistan and Algeria) account for over 90% of the total cases globally ⁴.

Malaria is a parasitic disease infecting humans and is transmitted by female mosquitoes of the genus Anopheles ⁵. Plasmodium species are the cause of human malaria deaths which were 450,000 with 218 million cases in 2017 and 228 million in 2018 ⁶. Africa still holds a high proportion of the global malaria burden recording 96% malaria cases and 94% of malaria deaths in global statistics ⁷.

The balance between Th₁ and Th₂ influences disease resistance and susceptibility in mice infected with L. donovani. Further studies have demonstrated that cytokines induced by Th₁ responses are linked to efficacious leishmaniasis control while Th₂ induced cytokines are associated with disease severity ⁸. Interferon γ (IFN-γ), during malaria and leishmanial infections, is confederated with protective immunity ⁹.

Co-infections of malaria and VL are the leading cause of increased mortality and morbidity associated with parasitic infections globally ^{4, 8}. However, regions where co-morbidity occurs, the impact in public health and the outcome of both diseases is poorly documented. Both infections present similar clinical signs and symptoms and many challenges remain to be overcome among patients living in co-endemic areas. Moreover, in human studies due to co-infection, the immune responses and the role they play in reduction of disease severity and protection is not clearly elucidated. High morbidity and mortality associated with the two diseases warranted us to study the disease outcome during a co-infection. Furthermore, it is unclear whether a co-infection could interfere with the clinical outcomes of the diseases. To better understand the clinical and immunological manifestation of a co-infection, we designed the present study to further elucidate the parameters and the effect they have in disease outcome in BALB/c mouse model.

2. Methodology

The present study involved a total of 82 BALB/c mice of either sex aged 6-8 weeks which were acquired from the Rodent facility, Animal Science Department at the Institute of Primate Research (IPR [www.primateresearch.org]), Karen, Nairobi, Kenya and housed in the same premises throughout the experimental period. The mice were housed in cages, the room was at an ambient temperature of 22C and relative humidity of 50-70%. They were fed on mice pellets (Unga Farm Care, Nairobi, Kenya) and water was provided ad libitum. The BALB/c mice were divided into 4 groups as follows: L. donovani- only infected mice (n= 25); L. donovani- P. berghei co-infected mice (n=22) (which were co-infected with P. berghei 60 days post-L. donovani infection); P. berghei-only infected mice (n= 25) and naïve (n=10) (which were given PBS only) (Figure 1). All infections were done intraperitoneally with 1 x 10⁶ P. berghei and/ or L. donovani parasites suspended in 100μl of PBS. Leishmania donovani inoculation was termed day -60 while P. berghei inoculation was day 0. Body weight measurements were done every day throughout the co- infection/ experimental period. Following co-infection, 3 mice were sacrificed on day 0, 4 and daily from the 6^th day till the end of the experimental period from both co-infected and single infected mice groups to obtain blood and spleen samples. Whole blood was used to prepare serum for IgG, IFN-γ and IL-4 quantification while tail prick blood samples which were done daily were used for parasitemia determination in P. berghei groups. Spleen samples were obtained in all L. donovani groups to prepare splenic impression smears for evaluation of L. donovani parasite load. In the survivorship experiment, BALB/c mice were euthanized using 3L/min Carbon IV Oxide (CO₂) for 2 minutes when they appeared moribund and date recorded. Spleen, liver and brain tissues were harvested on the 9^th day from both experimental and control groups for histological analysis. The experimental protocols and procedures were approved by the Institutional Science and Ethics Committee (ISERC) of the Institute of Primate Research (study ISERC/10/2016) and carried out according to its guidelines for Animal care and Handling.

Leishmania donovani strain NLB-065 was obtained from hamster’s spleen by aspirate and cultured in complete Schneider’s Insect media (Sigma-Aldrich Laborchemikalien GmbH, Seelze, Germany). Promastigotes at the stationary phase were harvested by centrifugation at 280g at 4°C for 15 minutes as described ¹¹. The pellet was later washed in sterile Phosphate Buffered Saline (PBS) by centrifugation as before. These parasites were then used to infect mice and for Soluble Leishmania antigen (SLA) preparation.

Cryopreserved stocks of Plasmodium berghei ANKA (wild type, supplied by Malaria Research and Reference Reagent Resource Center program (MR4)) were retrieved from liquid nitrogen and revived by thawing at 37°C in a water bath followed by three washes in PBS at 250g rpm for 10 minutes. The parasites were used to infect the BALB/c mice and for malaria crude antigen preparation used in antibody quantification using ELISA.

The pellet of harvested promastigotes was enumerated and 1 x 10⁸ promastigotes suspended in 2ml PBS, followed by 3 cycles of freeze-thawing in liquid nitrogen and 4 sonication cycles of 18 AMP on ice for 20 seconds each. The parasite suspension was later centrifuged at 10,000g for 30 minutes at 4°C. The supernatant was collected and protein concentration quantified using a Bio-Rad protein assay kit (BioRad Laboratories, USA) and later stored at -80°C until use.

Plasmodium berghei antigen was prepared from parasitized Red Blood Cells (pRBCs) obtained from BALB/c mice ¹². Four ml of heparinized blood was washed 3 times with PBS at 2000 rpm for 6 minutes. One ml of 0.1 % saponin in PBS was added to the pellet to lyse pRBCs and incubated at room temperature for 10 minutes with occasional mixing. Eight ml of PBS were added and centrifuged at 330g for 30 minutes at 4°C. The supernatant and RBC ghost were removed and the lysate washed 3 more times until it was dark red. The suspension was sonicated 6 times for 10 seconds with a 1-minute interval in ice to lyse parasite cell wall. The lysate was transferred to microcentrifuge tubes and centrifuged at 1,500g for 60 minutes at 4°C. The supernatant, which was the antigen, was harvested and filter sterilized. Protein concentration was determined using the Bio-Rad kit (BioRad Laboratories, USA).

The body weights were measured for both the experimental and control groups daily using a digital laboratory weighing balance (Amazon, USA) and results recorded in grams.

For P. berghei infected BALB/c mice parasite density was determined by examining a Giemsa-stained thin smear prepared from a tail blood prick, daily beginning on day 4 post-P. berghei infection. Parasitaemia was determined at 100X oil immersion under a microscope. Uninfected and infected red blood cells were identified and counted from different fields of view. About 500 erythrocytes were counted per slide and parasitemia was calculated as described ¹³. For L. donovani amastigotes, splenic impression smears from sacrificed mice were prepared. The fixing of slides and staining procedures was similar to the preparation of P. berghei for parasitemia above. Parasites were counted per 500 splenic cell nuclei at 100X oil immersion under a microscope ¹⁴.

Anti-parasite antibodies were performed as described ¹¹. Briefly, polystyrene Micro-ELISA plates (Nunc, Copenhagen, Denmark) were coated overnight with 100 μl per well of antigens at a concentration of 10 μg/ml and 5μg/ml for SLA and malaria crude antigen respectively in PBS pH 9.6. The plates were washed 5 times with 200 μl/well 0.05% Tween₂₀ in PBS (washing buffer) to remove excess antigen and later blocked with 200 μl/ well of 1% bovine serum albumin (BSA) in washing buffer and incubated at room temperature for 1 hour. The plates were washed 5 times as above to remove excess blocking buffer and 100 μl of the serum samples were added to the wells and the plate incubated at room temperature for 2 hours. The plates were washed again 5 times with the washing buffer to discard unbound serum and 100 μl of horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma- Aldrich, USA) diluted at 1:2000 in 1% BSA in washing buffer was added to each well followed by 1 hour incubation at room temperature. The plates were washed 5 times as before and 100 μl of Tetramethylbenzidine ((TMB), ThermoFisher, USA) substrate added to each well. Optical densities were read at 630 nm using an ELISA reader (Dynatech Laboratories, Sussex, UK) after 15-minute incubation in the dark.

Polystyrene micro- ELISA plates were coated overnight at 4°C with 100μl of mAb AN 18 and mAb 11B11 monoclonal antibodies specific for IFN-γ and IL-4 respectively on separate plates. Bovine serum albumin (0.1%) in PBS/0.05% Tween₂₀ buffer (washing buffer) was used to block nonspecific binding sites for 1 hour at 37°C. The plates were washed 3 times with washing buffer before the addition of 100 μl of serum samples and mouse cytokine standards and incubated for 2 hours at room temperature. Hundred microlitres of monoclonal cytokines detecting antibody, R4-6A2- biotin and BVD6-24G2 (0.5 µg/ml) were added per well and the plate incubated for 1 hour at room temperature. The plates were washed 5 times as before and 100μl Streptavidin (1:1000) added to each well before incubation at room temperature for 1 hour. Unbound Streptavidin was discarded by washing the plates 5 times and 100μl of TMB peroxidase substrate added to each well. The plates were incubated for 30 minutes in the dark and optical densities read at 630nm in a microplate reader (Dynatech Laboratories, UK).

Following the challenge of BALB/c mice with L. donovani and/ or P. berghei in the respective groups, four mice from each group were used for the determination of survivorship ¹⁵. A survivorship curve was plotted from the percentage of the number of surviving mice for 15 days.

Following harvesting of the spleen, liver and brain tissues of one mouse from each group on the 9^th-day post-infection, the organs were fixed in 10% formalin. They were later processed as described ¹⁶. Briefly, the tissues were dehydrated in alcohol at increasing concentrations for 1 hour before being immersed in xylene and embedded in melted paraffin wax for 3 hours. The sections were later cut at 4μm thick using a microtome and mounted on microscope slides where they were stained with hematoxylin-eosin stain (Abcam, USA). After they had air-dried, they were viewed under a light microscope at X400 and X100 magnifications and micrographs of the slides were captured.

Statistical analysis was conducted using GraphPad prism software package version (8.01). Body weight, P. berghei parasitemia, L. donovani parasite load, their respective IgG, IFN-γ and IL-4 responses were calculated as mean ± standard error of the mean (SEM). Mean body weights were compared using a one-way analysis of variance (ANOVA) and adjusted with a Tukey post hoc test. Intragroup statistical analysis was tested using two- way ANOVA while independent t-test was used to analyse intergroup means. For correlation analysis, Spearman’s rank correlation test was used. Kaplan- Meier curve was utilized in comparing the different survivorship rates. A difference was considered significant when the P-value was less than 0.05 (P < 0.05).

This study and all experimental protocols were approved by the Institutional Science and Ethics Committee (ISERC) of the IPR, Karen, Nairobi, Kenya (study ISERC/10/2016), whose membership is constituted based on guidelines issued by the World Health Organization for committees that review biomedical research, by the NIH, by PVEN, and by the Helsinki Convention on the Humane Treatment of Animals for Scientific Purposes. The IRC-ACUC is nationally registered by the National Commission for Science, Technology, and Innovation, Kenya.

3. Results

Following co-infection, variations in body weight were observed throughout the experimental period. Leishmania donovani- only and L. donovani- P. berghei co-infected mice group had considerable higher body weight as they were inoculated with L. donovani parasites 60 days prior to co-infection period. Body weight changes in the co-infected and control groups at the termination date ranged from (30.415-21.22g) which was not significantly different (F= 0.8427; P= 0.5402). The mean body weight of L. donovani- P. berghei co-infected mice group ranged from 32.4g on day 0 to 28g on the 10^th day which showed a significant decrease (P=0.0001). There was a slight drop in the mean body weight of L. donovani- only infected mice group from 32.2g on day 0 to 30.4g at the end of the experimental period (P=0.0001). Plasmodium berghei- only infected mice group had a slight decrease in body weight from 22.55g on day 0 to 21.58g on the 9^th day when the last BALB/c mouse appeared moribund and was sacrificed (P=0.0001). There was however an insignificant increase in mean body weight in the naïve mice group from 22.72g to 23.27g at the end of the experimental period (P=0.710, Figure 2).

Upon evaluating L. donovani parasite load and P. berghei parasitemia, there was a significant difference in the mean parasite load in L. donovani- P. berghei co-infected and L. donovani- only infected BALB/c mice groups (F=26.65; P= 0.0021, Figure 3) and mean P. berghei parasitemia between L. donovani- P. berghei co-infected and P. berghei- only infected BALB/c mice (F= 16.35; P=0.0099, Figure 4). The mean splenic amastigote load in L. donovani- P. berghei co-infected mice group decreased from 9.85% on day 0 of co-infection to 9.56% at the end of the experimental period (P<0.0001) while the mean L. donovani parasite load in L. donovani- only infected mice was 9.85% on day 0 and 17.84% at the end of the experimental period (P<0.0001) hence remarkably different.

The mean P. berghei parasitemia in L. donovani- P. berghei co-infected mice group was initially 0% on day 4 of co-infection but increased steadily to 14.88% at the end of experimental period (P = 0.0345). Plasmodium berghei- only infected mice had a mean parasitemia of 2.82% on day 4 and on day 9 when the last BALB/c mice appeared moribund and were sacrificed, the parasitemia had markedly increased to 23.45% (P= 0.0222).

Following the quantification of total IgG specific responses in mice by ELISA, there was no significant difference in the mean Leishmania specific optical density (OD) in L. donovani- P. berghei co-infected and L. donovani- only infected BALB/c mice groups (F= 2.881; P= 0.1118). Mean Leishmania specific optical density (OD) compared in L. donovani- P. berghei co-infected BALB/c mice were 0.8873 on day 0 and 1.0287 at the end of experimental period (P< 0.0001, Figure 3). The mean OD level in Leishmania- only infected mice group ranged from 0.8873 at the beginning of the co-infection period to 1.2597 at the end of the experimental period (P< 0.0001).

Data analysis revealed that the mean Plasmodium specific IgG OD levels in L. donovani- P. berghei co-infected mice were almost as twice higher as compared to P. berghei- only infected BALB/c mice and tis was significantly different (F= 4.436; P= 0.0463). The mean Plasmodium specific IgG OD levels in L. donovani- P. berghei co-infected BALB/c mice group had 0.033 OD at day 0 and 1.285 OD at the end of experimental period (P= 0.002, Figure 4) while mean OD for P. berghei only infected mice ranged from OD of 0.032 on day 0 and 0.85 at the end of experimental period (P= 0.0002).

The relationship between antibody responses and parasite burden was assessed. Data analysis indicated that an increase in P. berghei parasitemia coincided with an increase in L. donovani specific IgG antibodies in (F= 2.301; P= 0.0401) but an increase in L. donovani parasite load did not correlate with an increase in P. berghei IgG antibody responses in L. donovani- P. berghei co-infected mice (F= 0.1057; P= 0.4764). Following infection with both L. donovani and P. berghei, results indicated a decrease in L. donovani parasite load coincided with a decrease in L. donovani IgG OD values which declined at day 0 post-co-infection to day 6. In general, it was noted that an increase in parasite load resulted in a net increase in OD values as shown from day 7 of co-infection to the end of the experimental period. Similar results were seen in L. donovani- only infected mice groups where a decrease in parasite load resulted in a decrease in OD values and vice-versa however these results were not significant ((r= 0.01130 and 0.1816 respectively) P= 0.5345, Figure 3).

An increase in parasitemia also coincided with a peak in P. berghei IgG antibody responses. High OD values observed in L. donovani- P. berghei co-infected mice group resulted in high parasitemia while low IgG responses in P. berghei only infected mice group resulted in low parasitemia. A moderate correlation in the mean parasitemia in L. donovani- P. berghei and P. berghei- only infected mice groups with their respective IgG antibody responses were observed ((r= 0.5919 and 0.4833 respectively) P= 0.05, Figure 4).

Interferon gamma (IFN-γ) responses of L. donovani and P. berghei were quantified by measuring ODs by ELISA. We ascertained a decrease in circulating IFN-γ responses in L. donovani- P. berghei on day 4 post co- infection before increasing steadily to 0.8653 OD on day 8. Mean OD started to decrease significantly to 0.4229 on day 10 of co-infection (P= 0.0012). There was a steady decrease in IFN-γ responses in L. donovani only (P= 0.0002) while P. berghei only infected mice registered a peak in mean IFN-γ levels on day 6 of co-infection after which it began to notably decrease to 0.5264 OD (P= 0.0034). Results also indicated a notable difference in circulating IFN-γ responses within the groups during the co-infection period (P<0.0001, Figure 5).

When we assessed IL-4 responses and it was noted there was a decrease in IL-4 levels from day 0 to day 6 post co-infection. The mean OD levels increased significantly to a high OD of 0.6914 at the end of the experimental period (P= 0.0001). We also noted a steady significant increase in circulating IL-4 levels in L. donovani only (P= 0.0013) and P. berghei only (P= 0.0079) infected mice during the experimental period. Data analysis revealed that IL-4 levels were significantly different between the experimental and control groups during the experimental period (P<0.0001, Figure 5).

The impact of dual and single infections on mice survival was assessed. There were significant differences in the survivorship rate of BALB/c mice in experimental and control groups (P= 0.05, Figure 6). The L. donovani- P. berghei infected mice survived longer than the P. berghei only mice group while L. donovani only and naïve mice groups continued to survive up to the 20^th day when they were censored. On day 9 post-co-infection, 75% of the L. donovani- P. berghei co-infected mice had survived and each mouse was sacrificed on the 10^th, 12^th and 13^th day. None of the L. donovani only infected BALB/c mice succumbed to infection within the experimental period while 50% of P. berghei only infected BALB/c mice were sacrificed on the 6^th day and the remaining two were sacrificed on the 7^th and 9^th day.

Histological analyses were undertaken on the 9^th day of the co-infection period to assess the structural changes of the spleen, liver and brain tissues from all the groups. Naïve tissue sections of each organ were also included. The micrographs were viewed at X100 and X400 under a light microscope. Hematoxylin and Eosin-stained micrograph of the spleen showed an enlargement of red and white pulp elements accompanied by a loss in the typical structure of the germinal center with even distribution of granulomas and hyaline deposits in L. donovani- only mice which were more evident than L. donovani- P. berghei co-infected BALB/c mice. Massive sequestration of parasitized erythrocytes and accumulation of black to dark brown hemozoin which was disseminated in the red pulp was more pronounced in P. berghei- only infected mice as compared to L. donovani- P. berghei co-infected mice group (Figure 7).

H&E- stained liver section revealed lymphocytosis with hyaline deposits and granulomas in Leishmania only infected BALB/c mice. Presence of granulomas, necrosis as a result of hepatocyte injury and congestion of hepatic portal vessels was observed in L. donovani- P. berghei infected mouse. Multifocal lymphocytes and congestion of blood vessels was evident in P. berghei only infected mouse. No histopathological changes were present in the naïve control mice (Figure 8).

H & E- stained sections of the brain were assessed and P. berghei only infected mice group showed microhemorrhages and massive leukocyte infiltration were observed while few leukocytes in L. donovani- P. berghei co-infected mice were seen. No morphological changes were observed in Leishmania- only and naïve control mice (Figure 9).

4. Discussion

The present study was an attempt to evaluate clinical and immunological parameters of Leishmania donovani and Plasmodium berghei co-infection aimed at establishing the disease outcome in BALB/c mouse model.

There was an overall reduction in body weight in all the infected mice as compared to the non- infected naïve controls. Leishmania donovani infected mice had considerable higher body weight as compared to P. berghei and naïve mice groups as it took 60 days to establish L. donovani infection and 4 days for P. berghei infection and the standard protocol was to use mice aged 6- 8 weeks. In separate studies, ^{16, 17} associated diminution in body weight to the loss of appetite, reduced metabolism, altered gut function, hypoglycemia and most importantly massive destruction of erythrocytes. In addition, P. berghei infected rats ¹⁹ and L. donovani infected mice ²⁰ studies reported high anemia which was due to the destruction of erythrocytes and splenocytes respectively.

In our study, we established pre-infection with L. donovani caused a delay and low levels of P. berghei parasitemia in the onset of the malaria infection which was accompanied with a reduction in L. donovani load. However, during chronic P. berghei infection, the L. donovani load in co-infected mice accelerated, differently to L. donovani- only infected mice, which may suggest malaria may predispose an increased L. donovani infection. A decline in L. donovani parasite load during the early phase of co-infection in L. donovani- P. berghei co-infected mice group implies that superimposing P. berghei in L. donovani infection may contribute to a reduction in parasite load.

The current study found out that an increase in L. donovani parasite load resulted in high IgG antibodies. A study to quantify IgG responses in cutaneous leishmaniasis (CL) patients ²¹ reported high IgG antibody levels but this was found not to play any role on immunity. In the current study, reduction in IgG responses in co-infected mice which we observed may signify a lower parasite burden as compared to a single infected mice group.

Plasmodium berghei specific IgG plays a substantial role in mediating inhibition of pre-erythrocytic infection in malaria ²². As a result of this, it may explain the elevated IgG antibodies in mice infected with L. donovani- P. berghei co-infected as compared to P. berghei- only group mice. Previous research by ²³ has indicated high antibody titre correlates to high parasitemia levels which is the hallmark in protection against P. berghei.

It is documented that erythrocytic stage of malaria infection triggers potent IFN-γ responses in both human ²⁴ and murine studies which explains an increase in IFN-γ responses in the co-infected group during the early phase of co-infection. During this period, we observed low L. donovani parasite load and P. berghei parasitemia levels suggesting some level of immune protection as a result of superimposed P. berghei on L. donovani infection. Previous studies revealed that pro-inflammatory cytokines such as IFN-γ, TNF-α and IL-12 secreted by Th1 subset play a significant role in effective control of L. donovani infection ²⁵. As malaria infection advanced in the co-infected group, we noticed an increase in anti-inflammatory cytokine (IL-4), a crucial regulatory cytokine secreted in mice during pathology for protection during acute phase of malaria ²⁶ accompanied with a decrease in pro-inflammatory cytokine (IFN-γ). An increase in secreted IL-4 correlated with a decrease in IFN-γ levels and subsequently serum IgG levels. Leishmania donovani parasite load and P. berghei parasitemia levels increased with an increase in serum IL-4 levels while IFN-γ reduced towards the end of the experimental period suggesting that Th2 induced cytokines are linked to disease severity. From this study, we demonstrated that the balance between Th1 and Th2 determines disease resistance and exacerbation during L. donovani and P. berghei co-infection.

In areas of co-endemicity, the population are continuously exposed to malaria and leishmaniasis bite hence suffer from possible recurrent infections. This leads to possibility of immunomodulatory effects that may result in interference in the outcome of the two diseases. In the current study, the co-infected group had a higher survival rate compared to P. berghei – only infected group. However, the L. donovani – only group survived the longest suggesting that L. donovani may have conferred some immune protection presumably due to high IFN-γ levels as compared to IL-4 in the mice group. In general, it was noted that a decline in IFN-γ and increase in IL-4 cytokines towards the chronic stage of infection correlated with an increase in L. donovani and/ or P. berghei parasite burden, considerable body weight loss and decrease in survivorship in all the experimental groups. The results were in agreement with other studies by ^{32, 33} show that mice infected with P. berghei only had a high mortality rate as compared to Leishmania only infected mice. In addition, it was reported that co-infection decreases the mortality rate during acute P. yoelii infection ²⁹.

The findings of the current study demonstrated the presence of functional granulomas in single and co-infected L. donovani mice previously done by ³⁰ which serve as a distinctive feature of hepatic resistance. In another study ³¹ showed that hyaline deposits at times tend to replace disrupted reactive lymphoid follicles when the disease advances. These findings are similar to other works done on characterizing histopathological changes in liver and spleen during canine leishmaniasis ³² in which structural changes such as perisplenitis, accelerated changes in leukocyte numbers and related granulomas were commonly observed in Leishmania models ³³. Microhaemorrhages observed during cerebral malaria in P. berghei only infected mice is presumably due to the rupture of small blood vessels in basal ganglia or subcortical white matter when parasite filled erythrocytes block the blood vessels ³⁴.

Based on the current results, we have demonstrated that it is possible for the two diseases to interact in the same individual. The study results indicate significant differences in body weight, parasitemia, L. donovani parasite load and IgG levels. However, Th1 represented by (IFN-γ) and Th2 immune responses represented by IL-4 were not significantly different. Malaria- VL co-infected group showed reduction in disease severity by 21% in malaria and 35.3% in VL during the early phase of the study. Our study therefore concluded that concomitant malaria contributes to disease exacerbation in VL cases as observed in high mortality rate. Nevertheless, the study recommends an integrated approach for malaria and VL screening program where malaria is co-endemic in order to promptly initiate antileishmanial and antimalarial treatments. The insights of the study also emphasize on the need to have a better understanding of both clinical and immunological parameters in co- infected individuals to assist policymakers, clinicians and other stakeholders to develop better strategies for improved disease outcomes.

Acknowledgments

We thank the Malaria Research and Reference Reagent Resource Center (MR4) for providing us with Plasmodium berghei ANKA parasites. The authors would like to acknowledge the staff at Rodent Facility, Animal Science Department at Institute of Primate Research (IPR).

Funding

This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

Conceptualization, planned the study and designed the experiments: RMA, JMM, JCM and LO. Performed the assays: RMA and JCM. Analysed histopathology slides: DL. Analyzed the data and wrote the manuscript: RMA, JMM, JCM and LO.

References