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American Journal of Public Health Research
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The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
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Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
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Anne Lanham, Lillian Mwanri

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Schistosoma mansoni Co-infection Decelerates Murine Plasmodium berghei ANKA Induced Inflammatory Response and Organ Damage

Andrew Kasiti Muganda*, Edward Owiti Okonjo, James Nyabuga Nyariki, Dorcas Syokui Yole

Department of Applied and Technical Biology, Technical University of Kenya, Nairobi, Kenya

Abstract: Malaria is a severe infection caused by the Plasmodium parasite. It causes high mortality and morbidity, especially in the malaria endemic region. Schistosomiasis is caused by blood flukes and is the second leading parasitic infection after malaria in morbidity and mortality rate. These two infections are co-endemic in many areas. Both parasites have definitive and intermediate hosts and each utilizes the host protein differently. Each utilizes the host protein differently. The objective of this study was to determine the outcome of chronic S. mansoni infection in the regulation of Plasmodium berghei ANKA (PbA) associated disease severity and pathological events in a mouse model. Mice were infected with 200 Schistosoma mansoni cercaria and later with 50000 PbA infected red blood cells. Parasitemia was monitored on a two-day interval, to track the infection levels. Furthermore, relative organ weight, and inflammatory markers were quantified at the end of the study and analyzed at a p = 0.05. Chronic S. mansoni infection suppressed PbA parasitemia. Meanwhile, co-infection with S. mansoni and PbA protected against schistosomiasis induced hepatosplenomegaly. Moreover, induction of both schistosomiasis and malaria abrogated PbA induced elevated levels of TNF-α and IFN-γ cytokines, associated with inflammation. Co-infection with S. mansoni and PbA enhanced PbA induced suppression of anti-inflammatory cytokine IL-10. Standard histopathological analysis revealed that when mice were infected with S. mansoni or PbA alone they had pronounced organ damage, which was assuaged by co-infection with both parasites. Findings from this study clearly reveals that co-infection with S. mansoni and PbA, significantly protects mice against either S. mansoni or PbA -driven inflammatory response and organ damage.

Keywords: schistosomiasis, malaria, co-infection, inflammation, hepatology

Received December 12, 2021; Revised January 17, 2022; Accepted January 25, 2022

Citation: Andrew Kasiti Muganda, Edward Owiti Okonjo, James Nyabuga Nyariki, Dorcas Syokui Yole, Schistosoma mansoni Co-infection Decelerates Murine Plasmodium berghei ANKA Induced Inflammatory Response and Organ Damage. American Journal of Infectious Diseases and Microbiology, 10 (2). 58-69. January 2022. doi:10.12691/ajidm-10-2-2

1. Introduction

Malaria is a life-threatening parasitic disease in many tropical and subtropical countries. It is caused by the protozoan Plasmodium that is transmitted to people via bites of infected female Anopheles mosquitoes [1]. Schistosomiasis also known as bilharzia or snail fever is an acute and chronic infection caused by parasitic worms called schistosomes. The Center for Disease Control (CDC) considers schistosomiasis a “Neglected Tropical Disease” and is the second most devastating parasitic disease after malaria [2]. Epidemiological studies indicate that malaria and schistosomiasis are co-endemic in many regions [3].

Occurrence of malaria or schistosomiasis, being caused by blood parasites, result in anemia mostly due to high parasite burden [4,5,6]. Mortality resulting from helminthic infections including schistosomiasis has been grossly underestimated in most developing countries [7]. Schistosomiasis causes loss of 10.4 million Disability Adjusted Life Years (DALYs). However, it causes lower mortality than the morbidity it is usually associated with. These include and not limited to hepatosplenomegaly, hematochezia, lasting pains, diarrhea [8]. Women and children that do domestic chores in infested water and inadequate hygiene are at risk and vulnerable to infection [2]. In 2019, malaria caused up to 67% mortality in children under the age of five. An estimate of 822000 children were born with low birthweights in 33 countries in the case of malaria infection during pregnancy [1]. Schistosomiasis co-infection with malaria in children influences the development of acquired immunity associated with the resistance or the pathology linked to schistosomiasis [3].

In their cellular interactions during infection, Plasmodium species have been known to export protein after translation into the host red blood cells (RBC) decreasing serum protein. Studies have shown that the exported protein into the host cells, contribute to the virulence of malaria by altering cell permeability, increasing rigidity, and cytoadherence properties [9,10,11]. On the other hand, schistosomes decrease proteins associated with essential life cycles such as the citric acid, fatty acid, and urea cycles. However, they increase the quantities of the protein associated with stress and structural components [12]. Proteins such as plasma cytokines and chemokine are important in immune response and immunopathology in malaria and schistosomiasis.

Immune response associated with malaria is elicited when an infected/ parasitized red blood cell activates dendritic cell to maturity. Once a dendritic cell’s pattern recognition receptors (PRR) recognize a malaria pathogen associated molecular pattern (PAMP), the parasite is phagocytosed and the dendritic cell is activated. This upregulates the expression of Major histocompatibility complex class II (MHCII) molecules. MHCII presents the parasite peptides to T helper cells, which release pro-inflammatory cytokines (TNF-α, IFN-γ). These cytokines activate macrophages to phagocytes infected red blood cells (iRBC) and in the process release nitric oxide and reactive oxygen species [13].

The initial inflammation that occurs in schistosomiasis is thought to be as a result of eggs with a spine passing, through the multilayered epithelium [14]. This inflammation results in production of pro-inflammatory cytokines such as IFN-γ and TNF-α. The cytokine that is abundantly secreted by the S. mansoni eggs is Interleukin 4 [14]. Overproduction of these cytokines and movement of the eggs with spines results into host tissue damage. Wiedemann et al. [15], highlighted that helminths utilizes T- helper cell 2 (Th2) immune response. Th2 cell releases cytokines that activates eosinophil, mast cells, B cells, goblet cell among others.

Individual occurrence of malaria or schistosomiasis, is associated with inflammation, and chronic pathology. However, there is limited information on the inflammatory responses and histological changes during co-infection. This study helps to advance knowledge on immunological response, and pathology, during co-infection and disease progression. Additionally, it generates critical information on pathology, inflammation, and immunological events associated with co-infection. The findings from this study provides crucial information in developing strategies for the management of schistosomiasis and malaria co-infection as a tool towards overall reduction of morbidity and mortality in the two infections. The aim of this study was to determine the outcome of chronic S. mansoni infection in the regulation of P. berghei ANKA associated immune response in a BALB/c mouse model.

2. Methodology

2.1. Experimental Animals and Ethical Statement

Six weeks old Female adult BALB/c mice (weighing between 20-30g) and Plasmodium berghei ANKA (PbA) were obtained from Institute of Primate Research (IPR), Karen Nairobi. The mice were housed in cages at the Animal House in Technical University of Kenya. The mice were fed on mice chow. Wood chippings were used as bedding material. Drinking water was provided ad libitum. All mice were left to acclimatize for two weeks. Biomphalaria pfeifferi snails infected with S. mansoni were obtained from Kenya Medical Research Institute (KEMRI). This research was conducted in accordance with the internationally accepted principles for laboratory animal use and care as found in National Institutes of Health (NIH) guidelines.

2.2. Experimental Design

Forty-eight female BALB/c mice were assigned randomly into four groups with each group containing twelve mice. There were; Group 1: naive control mice, Group 2: S. mansoni infected mice, Group 3: S. mansoni- PbA co-infected mice and Group 4: PbA infected mice as indicated in Figure 1.

Figure 1. Experimental design: Showing the number of mice used and group distribution

2.3. Shedding of Cercaria

Biomphalaria pfeifferi snails were placed in an aquarium containing de-chlorinated water. The water was aerated. The snails were fed with clean dried lettuce. Dried oyster shells were placed as substratum and the water was changed twice a week. Snails were rinsed gently in de-chlorinated water to remove mucus which would cause shed cercariae to clump. The snails were placed in 250ml beaker with about 100ml of de-chlorinated water. The beaker was covered with transparent glass to prevent snails from crawling out and allow light to pass through. The beaker was illuminated with 100 watts electric lamp but taking care not to overheat the beaker. Cercarial suspension was poured in a clean beaker. The cercariae suspension was mixed and an aliquot of 50ul placed on a clean petri dish. The petri dish was placed on a dissecting microscope and cercariae observed at X10 objective. The number of cercariae per 50ul was counted after staining with Lugol’s iodine. Volume containing 200 cercariae was calculated.

2.4. Infection Procedures

Schistosoma mansoni: Mice were anaesthetized with intraperitoneal ketamine injection of 0.01ml ketamine per 20g-mouse. The mice were shaved on their lower abdomens. The mice were placed on an infection rack and a ring placed on the shaved area. Approximately 200 S. mansoni cercariae were placed in the ring.

Plasmodium berghei ANKA (PbA): Malaria parasites, transgenic Plasmodium berghei ANKA (PbA) stabilates had been stored in 10% glycerol at -196°C liquid nitrogen; in small capillary tubes of 2ml each. They were brought in a cryovial and left to thaw allowing time for the parasites to adapt to the normal temperatures before infection. Viability of the parasites in the capillary tube were checked by placing a drop of its contents on a slide. They were stained with giemsa and viewed under a microscope. EDTA saline glucose (ESG) buffer was used to dilute the parasites. Viability was checked again, before injecting 0.2ml of the diluted parasite into each donor mouse. On the ninth day of infection, parasitemia was checked by taking a drop of blood from the tail and examining under a microscope. The number of infected red blood cells (iRBC) was quantified.

No. of iRBC= Average parasitemia x 10^6 RBC/ml.

Serial dilution was done so as to get a solution of 5.0 x 10^4 iRBC/ml. To each experimental mouse, 0.2 ml of this dilution was injected intraperitoneally so that each mouse gets 5.0 x 10^4 iRBC. For co-infection group, P. berghei was inoculated only after S. mansoni infection had reached the chronic stage with and indicative S. mansoni eggs from the liver at week 7 post infection.

2.5. Parasitemia Determination

Thick blood smears were prepared from day 1 post-infection to gauge the patent period. Thin blood smears were prepared from mice tail and stained using 20% giemsa stain. Parasitemia was determined at x100 oil immersion under a microscope (Optica microscope).

2.6. Schistosoma mansoni Determination

Six weeks post-schistosomiasis infection, two mice, each per group were sacrificed and, perfused using phosphate buffered saline (PBS). Mesenteric vein was nipped and PBS injected into the heart of prilumbal surgically sectioned mice. Effluent containing adult worms was collected in a beaker and left to settle. The worms were observed at X20 magnification and images taken. Livers were observed for granuloma and images were taken. The livers were surgically removed and placed in petri dishes containing normal saline. The livers were gently crushed between glass plates and then homogenized using a blender. The homogenates were centrifuged at 1000 revolution per minute (RPM) for 3 minutes. Washing was done three times using normal saline discarding the supernatant. After the third wash, normal saline was added to the pellet and suspension filtered using a sieve. The filtrates were observed under X10 objective and images taken.

2.7. Euthanizing the Mice and Organ Assessment

The mice were sacrificed on the 7th days post-malaria infection. Prilumbal surgical sectioning of mice was performed to draw blood via cardiac puncture after the mice were anesthetized with 0.02ml ketamine per 30-gram mouse. Blood was placed in labelled vials. Spleen, brain, kidney, lung and liver were collected surgically and weights assessed using an analytical balance (Mettler PM34, DoltaRange). The relative organs weight of the normal control and those of the experimental groups were compared. Spleen, kidney, liver and brain were stored in 4% formalin for histopathology.

2.8. Preparation of Sera

Vials containing blood were left at room temperature for 3 hours then centrifuged for 5 minutes at 10,000 RPM to obtain serum and stored at 4°C for immunological assays (Centurion Scientific Ltd K240R, UK).

2.9. Determination of Cytokines Levels

Sandwich enzyme linked immune sorbent assay (ELISA) was used to determine serum cytokine levels of TNF-α, IFN-γ and IL-10. Invitrogen cytokines kit was used and the procedure was carried out as per manufacturer’s manual (Thermofischer Scientific, Califonia, USA). Quantitative reading at 450nm was done using ELISA optical reader (Multiscan ex-355, Thermo electron corporation, Waltham, Massachusetts, USA).

2.10. Histopathological Analysis

Standard histopathology examination of the kidney, spleen, liver and the brain were done to determine the extent of injury and whether co- infection offered organ protection. To achieve this, kidney, spleen, liver and the brain harvested were washed in phosphate buffered saline, fixed in 4% formalin, dehydrated in varying ethanol concentrations and embedded in paraffin. Prepared tissues were sectioned at 5µm then stained with Hematoxylin and Eosin for microscopy. The sections were examined under x40 objective lens for pathology and images taken using a compound microscope (Zeis Axio scope)

2.11. Data Analysis

Statistical analysis was done using GraphPad prism software package (Version 5.0). Images of sectioned organs were examined for pathology and images taken under a compound microscope (Zeis Axio scope). One-way ANOVA was done to compare the treatment groups with controls. For internal comparisons, Turkey’s post-hoc test was used. The results were given as a mean ± SEM with significance set at p<0.05.

3. Results

3.1. Effects of S. mansoni Infection on Liver Physiology

In this study, liver samples from the group of mice that were infected with S. mansoni were harvested, perfused and observed for the presence of granuloma (Figure 2A). Pictorial representation of the granuloma depicts the immunological response elicited by eggs lodged in the liver. Granuloma were characterized as white spots as indicated with blue arrows (Figure 2B). Also, the liver homogenates revealed the presence of eggs with lateral spine near the posterior end as shown with stars in Figure 2C.

Figure 2. Pictorial representation of adult worms (A), liver with granuloma (indicative black arrows; B) and eggs (at the star; C) a- group 2, b- group 3

3.2. Effects of S. mansoni Infection on the Parasitemia Levels of PbA

This experiment was done to determine if immune response induced during schistosomiasis would suppress or enhance the levels of PbA in blood. Levels of PbA in mice co-infected with S. mansoni and PbA was lower compared with the one infected with PbA alone though not statistically significant (p= 0.2039; Figure 3A & B). At the eighth day post infection, the percentage levels of parasitemia in the co-infected group of mice and PbA alone infected mice were 28.1% and 36.6% respectively (Figure 3A). The levels of parasitemia on the 8 days, was comparable in the two groups (Figure 3B).

Figure 3. Parasitemia in mice pre-infected with S. mansoni and the one infected with PbA alone. The analysis was conducted using student t-test, two tailed

3.3. Effects of PbA or S. mansoni Infection on Relative Organ Weight as a Marker of Organ Alteration

Infection with S. mansoni alone resulted in a significantly higher relative spleen (p<0.0003) and liver weights (p<0.0001) when compared with naive mice (Figure 4A &B). Mice infected with PbA alone or co-infected group of mice had significantly lower (p<0.05) spleen and liver relative organ weights compared to S. mansoni alone mice and comparable as naive mice (Figure 4A &B). The co-infected group of mice had similar spleen and liver weight relative to mice infected with PbA alone (Figure 4A &B). The relative brain weight for groups of mice infected with either S. mansoni alone or co-infected with S. mansoni- PbA were significantly reduced (p< 0.0001) in comparison to naive mice (Figure 4C). The brain of mice in the co-infected group were comparable to those from mice infected with S. mansoni and PbA alone (Figure 4C). The relative organ weights for the kidneys and lungs were comparable across all the infected groups (Figure 4D&E). However, mice from the co-infected group had marginally lower relative lung weight compared to S. mansoni and PbA infected mice (Figure 4E).

Figure 4. Relative organs weight post co- infection with PbA. Analysis was conducted using one-way ANOVA and Tukey’s post hoc test for comparisons. Indicated statistical difference *p<0.05, **p< 0.01, ***p< 0.001, ns- no significance

3.4. Effects of PbA Infection on Cytokines Level after Induction of Schistosomiasis

There was significant difference in the levels of TNF-α, IFN-γ and IL-10 in the experimental groups.

3.4.1. Co-infection of S. mansoni and PbA Dampened PbA Induced Elevated Levels of Pro-inflammatory Cytokines

The serum levels of parasite induced specific cytokines were analyzed to determine the role of PbA and/or S. mansoni in regulating inflammatory response. Infection with PbA alone resulted in significant elevations (p < 0.05) in the pro-inflammatory cytokine TNF-α and IFN-γ (Figure 5A & B). Notably, the levels of these pro-inflammatory cytokines were down regulated in co-infected group of mice. Co-infected mice had comparable levels of TNF-α and IFN-γ relative to mice infected with S. mansoni alone. Pro-inflammatory TNF-α and IFN-γ were significantly elevated (p < 0.05) in mice infected with PbA alone when compared to co-infection group of mice. Furthermore, PbA infection had significant elevation (p < 0.05) of the TNF-α and IFN-γ relative to S. mansoni infected mice. In contrast, mice infected with S. mansoni alone had significantly elevated levels (p < 0.05) of TNF-α relative to naive control mice (Figure 5A).

Figure 5. Effect of the infections on the levels pro-inflammatory cytokines in the study groups. Analysis was conducted using one-way ANOVA and Tukey’s post hoc test for comparisons. Indicated statistical difference *p<0.05, **p< 0.01, ***p< 0.001, ns- no significance

3.4.2. Co-infection of S. mansoni and PbA Induced Elevated Levels of IL-10

The levels of the anti-inflammatory cytokine interleukin-10 (IL-10) were significantly elevated (p= 0.0415) in both PbA and S. mansoni infected groups in comparison to naive control group (Figure 6). However, co-infected group of mice had marginal increase of IL-10 levels relative to mice infected with S. mansoni or PbA alone, though not statistically significant. The serum IL-10 levels remained marginally at the baseline when mice are infected with PbA or S. mansoni alone.

Figure 6. Effect of the infections on the levels anti-inflammatory cytokines in the study groups. Analysis was conducted using one-way ANOVA and Tukey’s post hoc test for comparisons. Indicated statistical difference *p<0.05, ns- no significance

3.4.3. Serum from Mice Co-infected with S. mansoni and PbA had Balanced Levels of Pro and Anti-inflammatory Cytokines

The ratios TNF-α: IL-10 and IFN-γ: IL-10 were calculated to determine if there was an imbalance between pro-inflammatory and anti-inflammatory cytokines during the infection period. The ratios of TNF: IL-10 (p =0.0008; Figure 7A) and IFN: IL-10 (p< 0.0001; Figure 7B) in mice infected with PbA alone were significantly higher relative to the ratio of naive control group, mice infected with S. mansoni alone and the co-infected group of mice. Co-infected group of mice had comparable TNF: IL-10 and IFN: IL-10 ratios relative to naive and group of mice infected with S. mansoni alone (Figure 7 A and B). Infection with S. mansoni alone or when co-infected with PbA significantly prevented the imbalance between the pro (TNF-α and IFN-γ) and anti-inflammatory (IL-10) cytokines (p<0.05; Figure 7 A and B).

Figure 7. Effect of the infections on the balance of pro-inflammatory and anti-inflammatory cytokines. Analysis was conducted using one-way ANOVA and Tukey’s post hoc test for comparisons. Indicated statistical difference *p<0.05, **p< 0.01, ***p< 0.0001, ns- no significance

3.5. Effects of PbA Infection on Organ Histology Following S. mansoni Infection as a Marker for Organ Damage

In the current study, chronic liver damage and necrosis in mice infected with S. mansoni alone was characterized by diffuse vascular congestion (C), multifocal areas of granulomatous reactions (G) as well as hepatocyte swelling in the focal area (S) (Figure 8b). Co-infected mice had mild liver injury characterized as focal areas of liver necrosis (star) and infiltration of the perivascular areas with mononuclear cells (arrowheads) (Figure 8c). Liver for mice infected with PbA had liver injury associated with focal area of vascular congestion (Figure 8d). Naive mice had a normal liver tissue (Figure 8a). There was mild injury in liver of co-infected mice compared to chronic liver damage in mice infected with S. mansoni alone. Mice infected with PbA alone were characterized with congestion only indicating a mild injury when compared to co-infected mice.

Figure 8. Comparison of the effect of the schistosomiasis and/ or malaria on the liver (×400, H&E). a- WT Naive, b- WT S. mansoni, c- WT S. mansoni- PbA, d- WT PbA

Kidneys of mice infected with schistosomiasis alone had kidney injury as the focal areas in the renal interstitium were infiltrated with mononuclear cells (arrow) and congestion (c) (Figure 9b). Co- infected mice kidneys had mononuclear cells which had infiltrated the focal area in the renal interstitium (arrow; Figure 9c). Kidney for mice infected with PbA (Figure 9d), and naive mice (Figure 9a) were normal at the end of the study period.

Figure 9. Comparison of the effect of the schistosomiasis and/ or malaria on the kidney (×400, H&E). a- WT Naive, b- WT S. mansoni, c- WT S. mansoni- PbA, d- WT PbA

Study mice infected with S. mansoni and/ or PbA as well as naive mice had normal spleen tissues as shown in Figure 10 a-d.

Figure 10. Comparison of the effect of the schistosomiasis and/ or malaria on the spleen (×400, H&E). a- WT Naive, b- WT S. mansoni, c- WT S. mansoni- PbA, d- WT PbA

Study mice infected with S. mansoni and/ or PbA as well as naive mice had normal brain tissues as shown in Figure 11a-d, despite the red pigmentation in in Figure 11b.

Figure 11. Comparison of the effect of the schistosomiasis and/ or malaria on the brain (×400, H&E). a- WT Naive, b- WT S. mansoni, c- WT S. mansoni- PbA, d- WT PbA

4. Discussion

Preliminary investigations were conducted to confirm if S. mansoni parasites had manifested in the chronic phase of infection before co- infecting mice with PbA. In the present study, there were limited differences in the intensity of the granuloma in the study groups, but their presence was a confirmation of schistosomiasis infection. In S. mansoni infection, granulomas usually cause destruction of the ova but result in fibrotic depositions in the host tissues, mostly in the intestines and liver [8,12]. Granulomas are normally characterized as white spots, are as a result of the eggs lodging in the liver and immune cells surrounding them. This granulomatous reaction tends to protect hepatocytes from further destruction from released toxins of the schistosomes eggs such as Schistosomes egg antigen (SEA) [14]. However, the intensity of granuloma is not directly associated with the infection level, adult worm after perfusion, or the number of eggs [16]. Immature eggs have an immunologically inert eggshell hence do not recruit macrophages around them to form granuloma but mature eggs secrete antigen that promote egg granuloma formation [17]. To overrule the assumption that granulomatous reaction might have been caused by another infection, the liver was perfused and adult schistosomes worms were observed. Eggs collected from the liver had lateral spines near the posterior end confirming S. mansoni infection which is accordance to WHO [8].

In the present study, parasitemia in mice pre-exposed to S. mansoni was slightly lower compared to mice infected with PbA alone though not statistically significant. Though the patent period was the same, it seemed like schistosomes counter Plasmodium effect hence the lower parasitemia. Several studies indicate that an underlying experimental schistosomiasis infection reduces the severity of subsequent malaria infection in co-infection studies [18,19]. Data from this study indicates that schistosomes protected the mice from severe malaria hence the marginally lower parasitemia compared to PbA alone infected mice though not statistically different (p= 0.2039). According to Donald et al. [18], the progression of malaria in the mice without schistosomes intervention is rapid compared to the group intervened with schistosomes. In a co-infection study of P. yoelli and S. mansoni, the latter reduced the malaria parasite levels in the liver This reduction led to fewer schizonts that would rupture into blood phase hence reduced parasitemia [20]. However, in a study conducted in Senegal, the low intensity occurrence of S. haematobium was found to lower the malaria effect whereas high schistosomiasis intensity promoted the occurrence of malaria [21]. This can be attributed to the different species of schistosome as compared to the current study.

In the current study, the relative weight of livers from mice infected with S. mansoni alone was elevated significantly as compared to naive mice. These findings concur with Egal, [22] and Lopes et al., [23] who reported a significant increase in liver of mice infected with schistosomes after 6 and 8 weeks that is attributed to hepatomegaly. In S. mansoni and PbA co-infected group of mice, there was a marginal increase relative to S. mansoni alone mice. This could be attributed to an antagonizing effect of malaria to schistosomiasis. Relative liver weight from co-infected group of mice and PbA alone mice was comparable to naive mice, as a result of antagonizing response of malaria on schistosomiasis. Most of these antagonistic effects elucidates that malaria parasite reduced the schistosomes effects in the host.

In this study, the spleen for mice infected with S. mansoni had a higher relative weight compared to naive mice. This was attributed to spleen enlargement. Additionally, co-infected group of mice had comparable spleen weight as PbA infected mice which was lower than naive mice. This indicates that P. berghei had an attenuating effect on the splenomegaly brought about by schistosomiasis. The spleen is known to clear the parasite, by removal of malaria parasite body from its red blood cell host as parasitized erythrocyte travel through the spleen [9,24]. Sequestering of splenocytes is a common occurrence in malaria infection [25] and hepatosplenomegaly is a common indicator in schistosomiasis as a result of retention of activated cells [8].

In the current finding the relative brain weight for mice infected with S. mansoni and co-infected group of mice was reduced when compared with naive mice. This finding confirms that schistosomiasis leads to destruction of brain and higher tropism for brain [26].

The relative kidney weights remained unaffected by S. mansoni and/or PbA in the current study. This is because the kidneys’ weight was marginally affected by these infections. In contrast, there was marginal increase in the size of kidney of children with malaria which worsened with increasing severity of morbidity as a result of malaria [27]. Schistosomiasis is known to cause kidney damage [7]. Whilst the current study supported the kidney injury; as mononuclear cells infiltrated foal area of renal interstitium, the relative kidney weight was not significantly affected.

In the present study, the relative lung weights remained unaffected by S. mansoni and/or PbA. These infections are known to marginally affect the lungs. In the lungs, chronic pulmonary schistosomiasis results in granuloma formation and fibrosis may lead to cor pulmonale [28]. In Plasmodium infection, pulmonary manifestation is part of severe systemic illness and its involvement has a lower frequency compared to the other complications [29]. However, there is limited information to depict if this complication could result in change of lung weight.

The cytokine levels in a host are a key indicator of infections and/ or activation of the host immune system to respond to infectious pathogens. In this study, levels of IFN- γ, TNF- α, and IL- 10 were measured in naive, S. mansoni, PbA, and co-infected group of mice. IFN- γ and TNF- α levels in PbA infected mice were significantly heightened relative to naive control mice. This finding are in agreement with a study done in Sudan by Barkat et al., [30] who reported that these pro-inflammatory cytokines in malaria infection are elevated more than in healthy control causing inflammation. IL- 10 levels had a statistically significant association with parasite levels in co-infected group of mice. Present data on co-infected group of mice is in agreement with other studies showing the correlation between parasitemia and IL-10 [30]. During a malaria infection, IL-10 is necessary for repressing hepatic pathology and suppressing the infection [5].

In the present study TNF- α, and IL- 10 were observed to be elevated in the group of mice infected with S. mansoni alone relative to naive mice. Furthermore, IFN-γ levels in S. mansoni infected mice was relative to naive mice. The finding corroborates with a study by Castro et al. [31], which indicated that IFN-γ levels are elevated in acute phase of schistosomiasis, as the levels of TNF- α and IL- 10 are elevated in chronic infection. However, in the co-infection study, TNF- α levels were slightly lower whereas IFN- γ, and IL- 10 were elevated relative to S. mansoni mice. Acute malaria elevates regulatory cytokines such as IL- 10 [32]. This is in agreement with our study and this explains the elevated levels in co-infected group of mice. IFN- γ levels in malaria infection are also elevated during the liver stage in hepatocytes [33]. This explains the lower levels in S. mansoni- PbA mice since schistosomes’ eggs are present in the liver with the protective schistosomes’ egg antigen (SEA) protein.

A review by Niikura et al. [5], indicates that high TNF- α /IL- 10 ratio is associated with severe malarial anemia. These results suggest that low IL-10 levels are associated with increased production of IFN-γ and TNF- α levels. This is contrary to our findings since the levels of IL- 10 were elevated in the S. mansoni and/or PbA infected mice relative to naive mice. The cytokine levels in schistosomes infected mice were higher than in naive mice. These observations are in agreement with a previous study by Meurs et al. [34], which describes that as Schistosoma infection intensity increases, Th1 cytokine responses decrease and the Th2 phenotype became more pronounced. This was exemplified by relatively higher IL-10 levels in co-infected group of mice and relatively lower IFN-γ, and TNF- α as compared to mice infected with PbA alone.

In the present study, the ratio of pro-inflammatory and anti-inflammatory cytokines was low in co-infected groups of mice relative to in S. mansoni or PbA or naive control mice. This suggests that the already established immune response by the primary infection by S. mansoni modulated the Th1 cytokine levels following the secondary infection by PbA. The down regulation of IFN- γ and TNF- α by IL- 10 was in line with a study by Montenegro et al. [35], on acute and chronic infections as low TNF- α and IL- 10 are associated with severe hepatic fibrosis and brought by chronic S. mansoni [36].

Histopathological analysis revealed an abnormal vascular congestion, hepatocyte swelling, granulomatous reactions characterized by spotty necrosis in the liver and kidney tubular necrosis with mononuclear cells infiltration in mice infected with S. mansoni and the co-infected group of mice. In the current study organs from P. berghei infected mice were marginally affected. These findings concur with a study on P. falciparum infection as the morphology of hepatocytes and endothelial cells in liver were generally unaffected [37]. Congestion of hepatocytes in this study can linked with the higher bilirubin in PbA infected mice. Liver cell necrosis was evident in PbA infected mice as the changes in liver histology is associated with total bilirubin [37]. Other organs in this study were visualized as normal for the PbA infected mice. These were contradicting findings which indicate lung damage [38], both liver and lung damage [39]. Brugat et al. [39], also indicates that malaria sequesters mostly in the liver, lungs and spleen and not Kidney and brain.

Mice infected with S. mansoni had granulomatous reactions in the liver, hepatocyte swelling and congestion. There was chronic liver damage in these mice as those co-infected with PbA had mild liver damage in this study. These findings confer with granulomatous responses seen in livers of rodents infected with S. mansoni [23,40]. Renal damage was evident in the mice infected with S. mansoni and the co-infected group of mice. The infiltration of mononuclear cells in the renal interstitium in both S. mansoni and co- infected group of mice signaled kidney damage in this study. Renal lesion is schistosomiasis indicates schistosomal glomerulopathy mostly in hosts with hepato- splenomegaly. These are characterized with type 1 membrane proliferation, glomerulonephritis and cellular proliferation [41]. Renal damage is associated with its functionality as Duarte et al., [42] highlighted this in S. mansoni infection. Brain and spleen were not affected in the current study though hemozoin could have altered the color of brain and the limited cell. Notably, organs damage induced by S. mansoni were reduced by occurrence of malaria in the co-infected group of mice.

5. Conclusion

The findings from this study clearly reveals that co-infection with S. mansoni and PbA, significantly protects mice against either S. mansoni or PbA-driven inflammatory response and organ damage. This is supported by; Chronic S. mansoni infection lowering PbA parasitemia of the host during co-infection. Both malaria and schistosomiasis cause inflammation though malaria causes higher inflammation as seen by the higher levels of pro-inflammatory cytokines. In co-infection, these are tentatively lower as anti-inflammation was higher in co-infection. The organ destruction assessment done showed that PbA had attenuating effect on S. mansoni damage especially on the liver and kidney

Data Availability

The data used to support the findings of this study are available from the corresponding author upon request.

Competing Interest Statement

The authors declare no conflict of interest.

Funding Statement

This work was self-funded with the help of supervisors, family, friends and well-wishers.

Acknowledgements

We thank the Institute of Primate Research (IPR), for providing us with Plasmodium berghei ANKA parasite. The authors would like to acknowledge Kenya Medical Research Institute (KEMRI), for providing us with snails infected with Schistosoma mansoni.

References

[1]   | WHO, World malaria report 2020- WHO. 2020.
[2]   | M. D. French et al., “Schistosomiasis in Africa: Improving strategies for long-term and sustainable morbidity control,” PLOS Neglected Tropical Diseases, pp. 1-6, 2020.
[3]   | T. O. Diallo et al., “Schistosomiasis Coinfection in Children Influences Acquired Immune Response against Plasmodium falciparum Malaria Antigens,” PLOS ONE, vol. 5, no. 9, pp. 1-7, 2010.
[4]   | K. R. Marr J.J., Nilsen T.W., Molecular Medical Parasitology, In Academic press London UK, vol. 66. 2012.
[5]   | M. Niikura, S. Inoue, and F. Kobayashi, “Role of Interleukin-10 in Malaria: Focusing on Coinfection with Lethal and Nonlethal Murine Malaria Parasites,” Hindawi Journal of Biomedicine and Biotechnology, vol. 2011, 2011.
[6]   | S. E. Butler et al., “Mechanism of anemia in Schistosoma mansoni-infected school children in Western Kenya,” American Journal of Tropical Medicine and Hygiene, vol. 87, no. 5, pp. 862–867, 2012, doi: 10.4269/ajtmh.2012.12-0248.
[7]   | “WHO | Schistosomiasis,” WHO, 2019.
[8]   | P. WHO, “Report: PAHO/ WHO Preparatory Meeting Elimination of Schistosomiasis Caribbean,” 2018.
[9]   | N. J. Spillman, J. R. Beck, and D. E. Goldberg, “Protein Export into Malaria Parasite – Infected Erythrocytes: Mechanisms and Functional Consequences,” Reviews in Advance, no. January, pp. 1-29, 2015.
[10]   | A. G. Maier, B. M. Cooke, A. F. Cowman, and L. Tilley, “Malaria parasite proteins that remodel the host erythrocyte,” Nature Reviews in microbiology, vol. 7, no. may, 2009.
[11]   | S. K. Prajapati, R. Culleton, and O. M. P. Singh, “Protein trafficking in Plasmodium falciparum- infected red cells and impact of the expansion of exported protein families,” Journal of Parasitology, pp. 1533-1543, 2014.
[12]   | M. Harvie, T. W. Jordan, A. Camille, and L. Flamme, “Differential Liver Protein Expression during Schistosomiasis,” Infection and Immunity, vol. 75, no. 2, pp. 736-744, 2007.
[13]   | M. F. Good, “Towards A Blood-Stage Vaccine for Malaria: Are We Following All The Leads?,” Nature reviews in Immunology, vol. 1, no. November, 2001.
[14]   | M. Abdulla, K. Lim, J. H. Mckerrow, and C. R. Caffrey, “Proteomic Identification of IPSE / alpha-1 as a Major Hepatotoxin Secreted by Schistosoma mansoni Eggs,” PLOS Neglected Tropical Diseases, vol. 5, no. 10, pp. 1-11, 2011.
[15]   | M. Wiedemann and D. Voehringer, “Immunomodulation and Immune Escape Strategies of Gastrointestinal Helminths and Schistosomes,” Frontiers in Immunology, vol. 11, no. September, pp. 1-13, 2020.
[16]   | P. W. Mola, I. O. Farah, T. M. Kariuki, M. Nyindo, R. E. Blanton, and C. L. King, “Cytokine Control of the Granulomatous Response in Schistosoma mansoni -Infected Baboons: Role of Exposure and Treatment,” American Society of Microbiology, vol. 67, no. 12, pp. 6565-6571, 1999.
[17]   | K. Takaki, G. Rinaldi, M. Berriman, A. Pagán, and L. Ramakrishnan, “Schistosoma mansoni eggs modulate the timing of granuloma formation to promote transmission,” Cell host and Microbe, pp. 1-10, 2020.
[18]   | N. D. Donald, M. Margaret, and O. Lucy, “The Role of Schistosoma mansoni Eggs in Protection against Plasmodium berghei Infected Mice,” Advances in Life Sciences and Technology, vol. 25, pp. 13-24, 2014.
[19]   | Mulei, “The effects of schistosomiasis and malaria co-infection on the clinical and pathological outcome in experimentally infected baboons (papio cynocephalus anubis),” 2012.
[20]   | T. Moriyasu, R. Nakamura, S. Deloer, and M. Senba, “Schistosoma mansoni infection suppresses the growth of Plasmodium yoelii parasites in the liver and reduces gametocyte infectivity to mosquitoes,” PLOS Neglected Tropical Diseases, pp. 1-17, 2018.

[21]  
J. Yves, L. Hesran, M. Cot, M. Lemaitre, and L. Watier, “Coinfection with Plasmodium falciparum and Schistosoma haematobium: Additional Evidence of the Protective Effect of Schistosomiasis on Malaria in Senegalese Children,” American Journal of Tropical Medicine and Hygiene, vol. 90, no. December 2003, pp. 329-334, 2014.

[22]   | R. Egal, “Effect of Different Durations of Schistosoma mansoni infection on the levels of some antioxidants in mice,” Infectious Diseases, (Auckl)., pp. 25-34, 2006.
[23]   | C. Lopes, V. Rc, V. Lr, B. Souza, and I. R. D. C. Rodrigues, “Histopathological study of Schistosoma mansoni infection in the murine model using the PC (Pará) and LILA (Maranhão) strains,” Mem Inst Oswaldo Cruz, vol. 101, pp. 273-277, 2006.
[24]   | D. A. Milner, “Malaria Pathogenesis,” Cold Spring Harbor Laboratory Press; Perspectives in Medicine, pp. 1-12, 2017.
[25]   | M. S. Id, A. Tempor, R. L. Id, and H. Nunes-cabac, “Trypanosoma brucei infection protects mice against malaria,” PLOS Pathogens, pp. 1-27, 2019.
[26]   | A. Pinto-almeida, T. Mendes, and R. N. De Oliveira, “Morphological Characteristics of Schistosoma mansoni PZQ-Resistant and -Susceptible Strains Are Different in Presence of Praziquantel,” Frontiers in Microbiology, vol. 7, no. April, pp. 1-11, 2016.
[27]   | O. M. Atalabi, A. E. Orimadegun, A. J. Adekanmi, and O. O. Akinyinka, “Ultrasonographic renal sizes, cortical thickness and volume in Nigerian children with acute falciparum malaria,” Malaria Journal, vol. 12, no. 1, pp. 1-7, 2013.
[28]   | “WHO | What is schistosomiasis?” https://www.who.int/schistosomiasis/disease/en/ (accessed Nov. 09, 2019).
[29]   | T. Niemann, H. P. Marti, S. H. Duhnsen, and G. Bongartz, “Pulmonary schistosomiasis-imaging features,” Journal of Radiology Case Reports, vol. 4, no. 9, pp. 37-43, 2010.
[30]   | W. R. J. Taylor, V. Cañon, and N. J. White, “Pulmonary manifestations of malaria: Recognition and management,” Treatments in Respiratory Medicine, vol. 5, no. 6. pp. 419-428, 2006.
[31]   | H. Barkat et al., “Prevalence of malaria and quantification of cytokine levels during infection in East Nile locality, Khartoum State: a cross-sectional study,” F1000Research, pp. 1-10, 2020.
[32]   | V. N. Castro, J. L. Rodrigues, D. T. Cardoso, and S. D. Resende, “Systemic Cytokine and Chemokine Profiles in Individuals with Schistosoma mansoni Infection and Low Parasite Burden,” Frontiers in immunology, vol. 9, no. December, pp. 1-12, 2018.
[33]   | L. Farrington et al., “Both inflammatory and regulatory cytokine responses to malaria are blunted with increasing age in highly exposed children,” Malaria Journal, pp. 1-11, 2017.
[34]   | R.K. Maheshwari, “The role of cytokines in malaria infection,” WHO Bulletin OMS, vol. 1, pp. 138-144, 1990.
[35]   | L. Meurs et al., “Cytokine Responses to Schistosoma mansoni and Schistosoma haematobium in Relation to Infection in a Co-endemic Focus in Northern Senegal,” PLOS Neglected Tropical Disease, vol. 8, no. 8, 2014.
[36]   | S. M. L. Montenegro et al., “Cytokine Production in Acute versus Chronic Human Schistosoma mansoni: The Cross-Regulatory Role of Interferon- gamma and Interleukin-10 in the Responses of Peripheral Blood Mononucleous Cells,” Journal of Infectious diseases, no. July, 2016.
[37]   | M. M. Mutengo et al., “Associated with Severe Hepatic Fibrosis in Schistosoma mansoni Chronically Exposed Individuals,” Hindawi Journal of Parasitology, vol. 2018, 2018.
[38]   | P. Viriyavejakul, V. Khachonsaksumet, and C. Punsawad, “Liver changes in severe Plasmodium falciparum malaria: histopathology, apoptosis and nuclear factor kappa B expression,” Malaria Journal, 2014.
[39]   | N. Valecha et al., “Case Report: Histopathology of Fatal Respiratory Distress Caused by Plasmodium vivax Malaria,” American Journal of Tropical Medicine and Hygiene, vol. 81, no. 2, pp. 758-762, 2009.
[40]   | T. Brugat et al., “Sequestration and histopathology in Plasmodium chabaudi malaria are influenced by the immune response in an organ-specific manner,” Cellular Microbiology, vol. 16, no. September 2013, pp. 687-700, 2014.
[41]   | B. Amaral, T. P. Silva, F. F. Dias, and F. M. Rosa, “Histological assessment of granulomas in natural and experimental Schistosoma mansoni infections using whole slide imaging,” PLOS ONE, pp. 1-20, 2017.
[42]   | 2 Geraldo Bezerra da Silva Junior1 and E. D. F. D., Daniella Bezerra Duarte, Elvino José Guardão Barros3, “Schistosomiasis-associated kidney disease: A review,” Asian Pacific Journal of Tropical Disease, vol. 3, no. 1, pp. 79-84, 2013.
[43]   | D. B. Duarte and L. A. Vanderlei, “Renal Function in Hepatosplenic Schistosomiasis – An Assessment of Renal Tubular Disorders,” PLOS ONE, pp. 1-15, 2014.