Schistosoma co-infection protects against brain pathology but does not prevent severe disease and death in a murine model of cerebral malaria

Co-infections of helminths and malaria parasites are common in human populations in most endemic areas. It has been suggested that concomitant helminth infections inhibit the control of malaria parasitemia but down-modulate severe malarial disease. We tested this hypothesis using a murine co-infection model of schistosomiasis and cerebral malaria. C57BL/6 mice were infected with Schistosoma mansoni and 8-9 weeks later, when Schistosoma infection was patent, mice were co-infected with Plasmodium berghei ANKA strain. We found that a concomitant Schistosoma infection increased parasitemia at the beginning of the P. berghei infection. It did not protect against P. berghei-induced weight loss and hypothermia, and P. berghei-mono-infected as well as S. mansoni-P. berghei-co-infected animals showed a high case fatality between days 6 and 8 of malarial infection. However, co-infection significantly reduced P. berghei-induced brain pathology. Over 40% of the S. mansoni-P. berghei-co-infected animals that died during this period were completely protected against haemorrhaging, plugging of blood vessels and infiltration, indicating that mortality in these animals was not related to cerebral disease. Schistosoma mansoni-P. berghei-co-infected mice had elevated plasma concentrations of IL-5 and IL-13 and on day 6 lower levels of IFN-γ, IL-10, monocyte chemoattractant protein-1 (MCP-1) and monokine induced by IFN-γ (MIG) than P. berghei-mono-infected mice. We conclude that in P. berghei infections, disease and early death are caused by distinct pathogenic mechanisms, which develop in parallel and are differentially influenced by the immune response to S. mansoni. This might explain why, in co-infected mice, death could be induced in the absence of brain pathology.     

Counter-regulatory anti-parasite cytokine responses during concurrent Plasmodium yoelii and intestinal helminth infections in mice

Malaria and helminth infections are two of the most prevalent parasitic diseases globally. While concomitant infection is common, mechanisms contributing to altered disease outcomes during co-infection remain poorly defined. We have previously reported exacerbation of normally non-lethal Plasmodium yoelii malaria in BALB/c mice chronically infected with the intestinal trematode Echinostoma caproni. The goal of the present studies was to determine the effect of helminth infection on IFN-γ and other key cytokines during malaria co-infection in the P. yoelii-E. caproni and P. yoelii-Heligmosomoides polygyrus model systems. Polyclonally stimulated spleen cells from both E. caproni- and H. polygyrus-infected mice produced significantly lower amounts of IFN-γ during P. yoelii co-infection than malaria-only infected mice. Furthermore, the magnitude of IFN-γ suppression was correlated with the relative amounts of IL-4 induced by these helminths (E. caproni = low; H. polygyrus = high), but not IL-10. Concurrent malaria infection also suppressed helminth-associated IL-4 responses, indicating that immunologic counter-regulation occurs during co-infection with malaria and intestinal helminths.     

Human helminth co-infection: No evidence of common genetic control of hookworm and Schistosoma mansoni infection intensity in a Brazilian community

Strong statistical associations between soil-transmitted helminths and schistosomes are frequently observed in co-endemic human populations, although the underlying explanations remain poorly understood. This study investigates the contribution of host genetics and domestic environment to hookworm and Schistosoma mansoni infection intensity and evaluates the role of genetic and non-genetic factors in co-variation of infection intensity. Detailed genealogical information allowed assignment of 1303 individuals living in the Brazilian community of Americaninhas, Minas Gerais state, to 25 pedigrees (containing between two and 1159 members) residing in 303 households. The prevalence of co-infection with both hookworms and schistosomes was high (38.5%), with significant correlation between Necator americanus and S. mansoni faecal egg counts. Bivariate variance component analysis demonstrated a modest but significant species-specific heritability for intensity of N. americanus (h² = 0.196) and S. mansoni infection (h² = 0.230). However, after accounting for demographic, socio-economic and household risk factors, no evidence for common genetic control of intensity of hookworm and schistosome infection was observed. There was some evidence for residual clustering within households but the majority (63%) of the covariance between N. americanus and S. mansoni infection intensity remained specific to the individual and could not be explained by shared genes, shared environment or other shared demographic, socio-economic or environmental risk factors. Our results emphasize the importance of exposure to hookworm and schistosome infection in driving the association between levels of infection with these species in hosts resident in areas of high transmission and suggest that much of this common exposure occurs outside the home.     

Plasmodium yoelii: Assessment of production and role of nitric oxide during the early stages of infection in susceptible and resistant mice

There is conflicting evidence regarding the role of nitric oxide (NO) in the process of resistance against blood-stage malaria parasites. In this study, we used two strains of mice infected with Plasmodium yoelii 17XL in order to assess the NO production profile and its possible role during the early stage of malaria infection. We found a greater elevation of NO production associated with a sharp increase in the levels of IFN-γ in infected DBA/2 mice, compared with infected BALB/c mice. This difference was associated with relatively lower parasitemia, a higher constituent ratio of infected reticulocytes, and greater survival in DBA/2 mice. Endogenous IFN-γ driving Th1 immunity was responsible for NO production. Moreover, schizonts treated in vitro with NO donors caused a delayed infection to BALB/c mice in a dose and time-dependent manner. These data, thus, suggest that NO may play an inhibitory role in Plasmodium infection.     

In vivo study of human Plasmodium knowlesi inMacaca fascicularis

Plasmodium knowlesi is a malaria parasite of Old World monkeys and is infectious to humans. In this study Macaca fascicularis was used as a model to understand the host response to P. knowlesi using parasitological and haematological parameters. Three M. fascicularis of either sex were experimentally infected with P. knowlesi erythrocytic parasites from humans. The pre-patent period for P. knowlesi infection in M. fascicularis ranged from seven to 14 days. The parasitemia observed was 13,686-24,202 parasites per μL of blood for asexual stage and 88-264 parasites per μL of blood for sexual stage. Periodicity analysis adopted from microfilaria periodicity technique of asexual stage showed that the parasitemia peak at 17:39 h while the sexual stage peaked at 02:36 h. Mathematical analysis of the data indicates that P. knowlesi gametocytes tend to display periodicity with a peak (24:00-06:00) that coincides with the peak biting activity (19:00-06:00) of the local vector, Anopheles latens. The morphology of P. knowlesi resembled P. falciparum in early trophozoite and P. malariae in late trophozoite. However, it may be distinguishable by observing the appliqué appearance of the cytoplasm and the chromatin lying inside the ring. Haematological analysis on macaques with knowlesi malaria showed clinical manifestations of hypoglycaemia, anaemia and hyperbilirubinemia. Gross examination of spleen and liver showed malaria pigments deposition in both organs.     

Plasmodium yoelii: Antibody and the maintenance of immunity in BALB/c mice

Subpatent persistence of parasitemia was detected for up to 7 weeks after infection of BALB/c mice with Plasmodium yoelii. Serum taken from recovered mice maintained parasitemias in recipient mice at a subpatent level when transferred repeatedly at 2-day intervals. Single doses of serum from convalescent donors delayed the course of infection in recipients. Small doses of transferred hyperimmune serum had the same effect, whereas large doses (>0.5 ml) totally suppressed parasitemia. Only a single secondary challenge of recovered mice was required in order to produce a maximally protective hyperimmune serum. Mice completely protected from a primary challenge with P. yoelii by transfer of hyperimmune serum were not at all resistant to a second challenge given some weeks later. After transfer of hyperimmune serum into mice with established P. yoelii infection, parasitemia fell to subpatent levels within 48 hr. During the first 21 hr after serum transfer, a progressive reduction in the proportion of ring forms present in blood smears was observed.     

Identification and differentiation of human schistosomes by polymerase chain reaction

Recent increasing number of travelers, immigrants and foreign workers from schistosomiasis endemic area has thus resulted in the importation of schistosomiasis to non-endemic countries. To avoid ova-induced pathogenicity, sensitive and specific diagnostic means at an early stage of infection are therefore crucial. In this study, we developed polymerase chain reaction (PCR) primers specific for human schistosome species. The PCR products were obtained in a species-specific manner (479 bp, Schistosoma mansoni; 365 bp, S. haematobium; 614 bp, S. japonicum; 303 bp, S. mekongi) and were detectable from 0.01 pg of total worm DNA (S. haematobium, S. japonicum, S. mekongi). The primer sets were also available for multiplex use. Although some difficulties were experienced in amplifying the parasite DNA from the infected animals, schistosome DNA could be detected from one day post infection. The PCR method described herein will therefore be beneficial to detect human schistosomiasis, after some improvements in this method.     

Metabolic alterations in the hamster co-infected with Schistosoma japonicum and Necator americanus

Co-infection with hookworm and schistosomes is a common phenomenon in sub-Saharan Africa, as well as in parts of South America and southeast Asia. As a first step towards understanding the metabolic response of a hookworm-schistosome co-infection in humans, we investigated the metabolic consequences of co-infection in an animal model, using a nuclear magnetic resonance (NMR)-based metabolic profiling technique, combined with multivariate statistical analysis. Urine and serum samples were obtained from hamsters experimentally infected with 250 Necator americanus infective L₃ and 100 Schistosoma japonicum cercariae simultaneously. In the co-infection model, similar worm burdens were observed as reported for single infection models, whereas metabolic profiles of co-infection represented a combination of the altered metabolite profiles induced by single infections with these two parasites. Consistent differences in metabolic profiles between the co-infected and non-infected control hamsters were observed from 4 weeks p.i. onwards. The predominant metabolic alterations in co-infected hamsters consisted of depletion of amino acids, tricarboxylic acid cycle intermediates (e.g. citrate and succinate) and glucose. Moreover, alterations of a series of gut microbial-related metabolites, such as decreased levels of hippurate, 3-hydroxyphenylpropionic acid, 4-hydroxyphenylpropionic acid and trimethylamine-N-oxide, and increased concentrations of 4-cresol glucuronide and phenylacetylglycine were associated with co-infection. Our results provide a first step towards understanding the metabolic response of an animal host to multiple parasitic infections.     

The effect of helminth co-infection on malaria in mice: a meta-analysis

The question of how helminths may alter the course of concurrent malaria infection has attracted much interest in recent years. In particular, it has been suggested that by creating an anti-inflammatory immune environment, helminth co-infection may dampen both protective and immunopathological responses to malaria parasites, thus altering malaria infection dynamics and disease severity. Both synergistic and antagonistic interactions are reported in the literature, and the causes of variation among studies are not well understood. Here, meta-analysis of 42 mouse co-infection experiments was used to address how helminths influence malaria parasite replication and host mortality, and explore the factors explaining variation in findings. Most notably, this analysis revealed contrasting effects of helminth co-infection in lethal and resolving malaria models. Whilst co-infection exacerbated mortality and increased peak parasitaemia in ordinarily resolving malaria infections (Plasmodium chabaudi and Plasmodium yoelii), effects among lethal malaria infections (Plasmodium berghei) tended to be in the opposite direction with no change in parasitaemia. In the subset of experiments on cerebral malaria models (P. berghei ANKA strain in a susceptible host), helminth co-infection significantly delayed death. These findings are consistent with the hypothesis that depending on the existing balance of pro- and anti-inflammatory responses mounted against malaria parasites in a given host, immune responses elicited by helminth co-infection may either promote or inhibit malarial disease. However, despite such broad patterns, a prominent feature of this dataset was great heterogeneity in effects across studies. A key future challenge therefore lies in explaining the biological causes of this variation, including a more thorough exploration of non-immunological mechanisms of helminth-malaria interaction.     

Leishsmania (Leishmania) amazonensis infection: Muscular involvement in BALB/c and C3H.HeN mice

Recent studies have provided some insights into Leishsmania (Leishmania) amazonensis muscular infection in dogs, although, muscular disease due to leishmaniasis has been poorly documented. The aim of our study was to evaluate involvement of Leishmania in muscular infection of two distinct mouse strains (BALB/c and C3H.He), with different genetic backgrounds. BALB/c mice, susceptible to Leishmania infection, showed, at the beginning of infection, a great number of infected macrophages among muscle fibers; however, in C3H.He resistant mice, muscle fibers were less damaged than in BALB/c mice, but some parasitized macrophages could be seen among them. A follow up of the infection showed an intense inflammatory infiltrate mainly composed of infected macrophages in BALB/c muscles and the presence of amastigotes within muscle fibers; while C3H.He mice exhibited a moderate inflammatory infiltrate among skeletal muscle fibers and an absence of amastigotes. Total destruction of muscles was observed in BALB/c mice in the late phase of infection (day 90) while C3H.He mice showed a process of muscle repair. We concluded that: (1) the muscles of BALB/c mice were more affected by leishmaniasis than those of C3/H.He mice; (2) Leishmania amastigotes are capable of infecting muscular fibers, as observed in BALB/c mice; (3) as inflammatory infiltrate is less intense in C3H.He mice these animals are capable of restoring muscular fibers.     

Inhibition of Plasmodium sporozoites infection by targeting the host cell

There is a great need of new drugs against malaria because of the increasing spread of parasite resistance against the most commonly used drugs in the field. We found that monensin, a common veterinary antibiotic, has a strong inhibitory effect in Plasmodium berghei and Plasmodium yoelii sporozoites hepatocyte infection in vitro. Infection of host cells by another apicomplexan parasite with a similar mechanism of host cell invasion, Toxoplasma tachyzoites, was also inhibited. Treatment of mice with monensin abrogates liver infection with P. berghei sporozoites in vivo. We also found that at low concentrations monensin inhibits the infection of Plasmodium sporozoites by rendering host cells resistant to infection, rather than having a direct effect on sporozoites. Monensin effect is targeted to the initial stages of parasite invasion of the host cell with little or no effect on development, suggesting that this antibiotic affects an essential host cell component that is required for Plasmodium sporozoite invasion.     

TLR9激动剂对约氏疟原虫体液免疫记忆的影响

为探讨TLR9激动剂对疟疾体液免疫记忆的影响,用非致死型约氏疟原虫感染BALB/c小鼠,感染前2 d注射TLR9激动剂CpGl826,90 d后进行二次感染。薄血膜染色法观察红细胞感染率,流式细胞术检测脾细胞悬液中记忆性和活化性B细胞百分比,双夹心ELISA法检测特异性抗体水平。结果显示,二次感染前,TLR9激动剂处理鼠记忆性和活化性B细胞以及抗体水平略高于对照组;二次感染后,其再感染发生率和虫血症水平均略低于对照组;活化性B细胞和抗体以及记忆性B细胞也分别于二次感染后1 d和3 d出现了有意义的升高,且升高幅度均略高于对照组。表明TLR9激动剂对约氏疟原虫感染后体液免疫记忆的建立和维持有一定促进作用。     

Metabolic profiling of a Schistosoma mansoni infection in mouse tissues using magic angle spinning-nuclear magnetic resonance spectroscopy

In order to enhance our understanding of physiological and pathological consequences of a patent Schistosoma mansoni infection in the mouse, we examined the metabolic responses of different tissue samples recovered from the host animal using a metabolic profiling strategy. Ten female NMRI mice were infected with ∼80 S. mansoni cercariae each, and 10 uninfected age- and sex-matched animals served as controls. At day 74 post infection (p.i.), mice were killed and jejunum, ileum, colon, liver, spleen and kidney samples were removed. We employed ¹H magic angle spinning-nuclear magnetic resonance spectroscopy to generate tissue-specific metabolic profiles. The spectral data were analyzed using multivariate modelling methods including an orthogonal signal corrected-projection to latent structure analysis and hierarchical principal component analysis to assess the differences and/or similarities in metabolic responses between infected and non-infected control mice. Most tissues obtained from S. mansoni-infected mice were characterized by high levels of amino acids, such as leucine, isoleucine, lysine, glutamine and asparagine. High levels of membrane phospholipid metabolites, including glycerophosphoryl choline and phosphoryl choline were found in the ileum, colon, liver and spleen of infected mice. Additionally, low levels of energy-related metabolites, including lipids, glucose and glycogen were observed in ileum, spleen and liver samples of infected mice. Energy-related metabolites in the jejunum, liver and renal medulla were found to be positively correlated with S. mansoni worm burden upon dissection. These findings show that a patent S. mansoni infection causes clear disruption of metabolism in a range of tissues at a molecular level, which can be interpreted in relation to the previously reported signature in a biofluid (i.e. urine), giving further evidence of the global effect of the infection.     

Schistosoma mansoni and Fasciola hepatica: Cross-resistance in mice with single-sex schistosome infections

Primary Schistosoma mansoni single-sex infections in mice, i.e., either male only or female only, did not stimulate any detectable level of heterologous resistance to challenge with Fasciola hepatica after 22 to 76 days, while statistically significant resistance to a challenge with F. hepatica was demonstrated in the presence of patent mixed-sex S. mansoni infections. Simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction in the number of schisto-some worms established, i.e., the burden being reduced by 40.1 and 43.9%, respectively. There was no reduction of the F. hepatica worm burden. Similar features could be observed with a time interval of 48 hr between the S. mansoni infection and the F. hepatica challenge, i.e., the schistosome burden being reduced by 34.2 and 45.6%, respectively. Furthermore, simultaneous infections with S. mansoni and F. hepatica induced a statistically significant reduction of the egg production capacity per paired female schistosome worm as compared with that of the S. mansoni control group. Tissue egg counts of the various intestinal sections were reduced by 92.8–99.6%.     

Penetration and maturation of Schistosoma mansoni in suckling and adult Swiss Webster and DBA/2 mice

Murine schistosomosis is a widely used experimental model of the human disease. Different methods have been employed to infect mice with Schistosoma mansoni cercariae, such as subcutaneous or intraperitoneal injections, the tail immersion technique, and the use of a metal ring placed on the abdominal skin of anaesthetized animals. An alternative method of infection that requires no anaesthesia and no restraint is to place suckling mice (10 days old) on a Petri dish with a small volume of water containing cercariae. In this study, we compared the penetration and maturation of S. mansoni in mice infected by this alternative method with those noted in mice infected at an older age (45 days) by the tail immersion technique. Besides evaluating the effects of age and method of infection, we also compared the susceptibility of two strains of mice (Swiss Webster (SW) and DBA/2). Mice were exposed to 100 cercariae and worms (by portal-hepatic perfusion) as well as eggs were recovered in the liver and intestines on postinfection (PI) days 35, 55 and 90. Skin penetration was very efficient (about 100%) irrespective of the mouse strain, sex, age and method of infection. Worm and egg recoveries were higher in SW mice at any PI interval, but strain differences tended to be less pronounced on PI day 90. In both strains, recoveries of worms and eggs were clearly higher in mice infected at a younger age (10 days old). This study thus suggests that infection of free-moving suckling mice is a suitable alternative to other methods of infection with S. mansoni.     

Schistosoma mansoni: Developmental arrest of miracidia treated with histone deacetylase inhibitors

In the present study, we examined the effect of the histone deacetylase (HDAC) inhibitors trichostatin A (TSA), valproic acid (VA) and sodium-butyrate on the metamorphosis of larvae of the human blood-fluke Schistosoma mansoni from the free-swimming miracidia into the intramolluskal sporocyst. We show that HDAC inhibitors block transformation in concentration dependant manner. TSA reversibly blocks this developmental process: only 13 ± 11% of TSA treated miracidia transform into sporocysts in-vitro, compared to 92 ± 3% in the mock-treated control. Other enzyme inhibitors such as cycloheximide or hydroxyurea had no effect on metamorphosis. For treatment of up to 4 h, the effect of TSA was completely reversible. Our data indicates that HDAC activity is necessary for the transformation of S. mansoni miracidia during infection of the snail host.     

Spatial heterogeneity of parasite co-infection: Determinants and geostatistical prediction at regional scales

Multiple parasite infections are widespread in the developing world and understanding their geographical distribution is important for spatial targeting of differing intervention packages. We investigated the spatial epidemiology of mono- and co-infection with helminth parasites in East Africa and developed a geostatistical model to predict infection risk. The data used for the analysis were taken from standardised school surveys of Schistosoma mansoni and hookworm (Ancylostoma duodenale/Necator americanus) carried out between 1999 and 2005 in East Africa. Prevalence of mono- and co-infection was modelled using satellite-derived environmental and demographic variables as potential predictors. A Bayesian multi-nominal geostatistical model was developed for each infection category for producing maps of predicted co-infection risk. We show that heterogeneities in co-infection with S. mansoni and hookworm are influenced primarily by the distribution of S. mansoni, rather than the distribution of hookworm, and that temperature, elevation and distance to large water bodies are reliable predictors of the spatial large-scale distribution of co-infection. On the basis of these results, we developed a validated geostatistical model of the distribution of co-infection at a scale that is relevant for planning regional disease control efforts that simultaneously target multiple parasite species.     

Cryptosporidium parvum: the course of Cryptosporidium parvum infection in C57BL/6 mice co-infected with the nematode Heligmosomoides bakeri

The effects of Heligmosomoides bakeri infection on the course of a concurrent Cryptosporidium parvum infection were studied in C57BL/6 mice. Mice were initially infected with 80 L₃ of H. bakeri and then challenged with 10⁴ oocysts of C. parvum, administered during the patent period of the nematode infection (28 day post H. bakeri infection). The number of C. parvum oocysts excreted in the feces and the number of adult H. bakeri in the small intestine were monitored during the experiment. Concurrent H. bakeri infection resulted in a prolonged course of infection with C. parvum. The intensities of both parasite infections were higher in co-infections. We also investigated the cellular immune response at 14 and 42 days post infection C. parvum. During infection with C. parvum there was an increase in production of IFN-γ and IL-12 but co-infection with H. bakeri inhibited IFN-γ secretion. The present study is the first to demonstrate that infection with H. bakeri markedly exacerbates the intensity of a concurrent C. parvum infection in laboratory mice and also affects immune effectors mechanisms in co-infection with H. bakeri.     

Plasmodium: Mammalian codon optimization of malaria plasmid DNA vaccines enhances antibody responses but not T cell responses nor protective immunity

We have evaluated the effect of mammalian codon optimization on the immunogenicity and protective efficacy of plasmid DNA vaccines encoding pre-erythrocytic stage Plasmodium falciparum and Plasmodium yoelii antigens in mice. Codon optimization significantly enhanced in vitro expression and in vivo antibody responses for P. falciparum circumsporozoite protein (PfCSP) and P. yoelii hepatocyte erythrocyte protein 17 kDa (PyHEP17) but not for P. yoelii circumsporozoite protein (PyCSP). Unexpectedly, more robust CD4⁺ and CD8⁺ T cell responses as measured by IFN-γ ELIspot, lymphoproliferation, and cytotoxic T lymphocyte assays were noted with native as compared with codon optimization constructs. Codon optimization also failed to enhance CD8⁺ T cell dependent protection against P. yoelii sporozoite challenge as measured by liver-stage parasite burden. These data demonstrate that the effect of mammalian codon optimization is antigen-dependent and may not be beneficial for vaccines designed to induce T cell dependent protective immunity in this malaria model.