Distinct placental malaria pathology caused by different Plasmodium berghei lines that fail to induce cerebral malaria in the C57Bl/6 mouse

ABSTRACT: BACKGROUND: Placental malaria (PM) is one major feature of malaria during pregnancy. A murine model of experimental PM using BALB/c mice infected with Plasmodium berghei ANKA was recently established, but there is need for additional PM models with different parasite/host combinations that allow to interrogate the involvement of specific host genetic factors in the placental inflammatory response to Plasmodium infection. METHODS: A mid-term infection protocol was used to test PM induction by three P. berghei parasite lines, derived from the K173, NK65 and ANKA strains of P. berghei that fail to induce cerebral malaria (CM) in the susceptible C57BL/6 mice. Parasitaemia course, pregnancy outcome and placenta pathology induced by the three parasite lines were compared. RESULTS: The three P. berghei lines were able to evoke severe PM pathology and poor pregnancy outcome features. The results indicate that parasite components required to induce PM are distinct from CM. Nevertheless, infection with parasites of the ANKADeltapm4 line, which lack expression of plasmepsin 4, displayed milder disease phenotypes associated with a strong innate immune response as compared to infections with NK65 and K173 parasites. CONCLUSIONS: Infection of pregnant C57BL/6 females with K173, NK65 and ANKADeltapm4 P. berghei parasites provide experimental systems to identify host molecular components involved in PM pathogenesis mechanisms.     

CTLA-4 blockade differentially influences the outcome of non-lethal and lethal Plasmodium yoelii infections

An immune response against malaria has to be tightly controlled. The production of pro-inflammatory cytokines is required to control parasites but the same cytokines are also involved in severe malaria. We have shown that CTLA-4 expression during Plasmodium berghei malaria dampens the immune response. This strain provokes a pro-inflammatory immune response that is associated with the pathology of cerebral malaria. Accordingly a blockade of CTLA-4 during the blood-stage of P. berghei malaria leads to an exacerbation of disease. To analyze the effects of a CTLA-4 blockade in a malaria model which is not prone to immune pathology we employed P. yoelii infection. Blood-stage infection led to a rapid induction of CTLA-4 on T cells. Using the non-lethal P. yoelii strain Py17NL we found that a blockade of CTLA-4 resulted in an increased T cell activation and IFN-gamma production, which was accompanied by a lower peak parasitemia and earlier parasite clearance. In contrast, blockade of CTLA-4 during infection with a P. yoelii strain exhibiting a higher parasitemia induced markedly increased serum-levels of TNF-alpha, which was associated with severe inflammation and reduced survival.     

Studies on enzyme variation in the murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi by starch gel electrophoresis.

Electrophoretic variation of the enzymes glucose phosphate isomerase, 6-phosphogluconate dehydrogenase, lactate dehydrogenase and glutamate dehydrogenase (NADP-dependent) has been studied in the African murine malaria parasites Plasmodium berghei, P. yoelii, P. vinckei and P. chabaudi and their subspecies. Horizontal starch gel electrophoresis was used throughout. The number of isolates examined in each subspecies varied from 1 (P. y. nigeriensis) to 24 (P. c. chabaudi). Extensive enzyme variation was found among isolates of most of the subspecies from which more than two such isolates were available for study. It is clear that the phenomenon of enzyme polymorphism is of common occurrence among malaria parasites. With the exception of P. berghei and P. yoelii, of which all isolates share an identical electrophoretic form of lactate dehydrogenase, no enzyme forms are shared between any of the 4 species of murine plasmodia. By contrast, within each species common enzyme forms are shared among each of the subspecies. The subspecies are nevertheless, distinguished from each other by the electrophoretic forms of at least one enzyme. The distribution and reassortment of enzyme variation among isolates of a single subspecies is in accordance with the concept of malaria parasites as sexually reproducing organisms. The study of variation among parasites present in individual wild-caught rodent hosts demonstrates that natural malarial infections usually comprise genetically heterogeneous populations of parasites. Nevertheless, the number of genetically distinct types of parasite of any one species present in a single infected host appears to be small. Generally not more than 2 or 3 clones of parasite of distinct genetic constitution are present in a single infected animal.     

The role of IL-18 in blood-stage immunity against murine malaria Plasmodium yoelii 265 and Plasmodium berghei ANKA

A possible protective role of IL-18 in host defense against blood-stage murine malarial infection was studied in BALB/c mice using a nonlethal strain, Plasmodium yoelii 265, and a lethal strain, Plasmodium berghei ANKA. Infection induced an increase in mRNA expression of IL-18, IL-12p40, IFN-gamma, and TNF-alpha in the case of P. yoelii 265 and an increase of IL-18, IL-12p40, and IFN-gamma in the case of P. berghei ANKA. The timing of mRNA expression of IL-18 in both cases was consistent with a role in the induction of IFN-gamma protein expression. Histological examination of spleen and liver tissues from infected controls treated with PBS showed poor cellular inflammatory reaction, massive necrosis, a large number of infected parasitized RBCs, and severe deposition of hemozoin pigment. In contrast, IL-18-treated infected mice showed massive infiltration of inflammatory cells consisting of mononuclear cells and Kupffer cells, decreased necrosis, and decreased deposition of the pigment hemozoin. Treatment with rIL-18 increased serum IFN-gamma levels in mice infected with both parasites, delayed onset of parasitemia, conferred a protective effect, and thus increased survival rate of infected mice. Administration of neutralizing anti-IL-18 Ab exacerbated infection, impaired host resistance and shortened the mean survival of mice infected with P. berghei ANKA. Furthermore, IL-18 knockout mice were more susceptible to P. berghei ANKA than were wild-type C57BL/6 mice. These data suggest that IL-18 plays a protective role in host defense by enhancing IFN-gamma production during blood-stage infection by murine malaria.     

Coinfection with nonlethal murine malaria parasites suppresses pathogenesis caused by Plasmodium berghei NK65

Mixed infection with different Plasmodium species is often observed in endemic areas, and the infection with benign malaria parasites such as Plasmodium vivax or P. malariae has been considered to reduce the risk of developing severe pathogenesis caused by P. falciparum. However, it is still unknown how disease severity is reduced in hosts during coinfection. In the present study, we investigated the influence of coinfection with nonlethal parasites, P. berghei XAT (Pb XAT) or P. yoelii 17X (Py 17X), on the outcome of P. berghei NK65 (Pb NK65) lethal infection, which caused high levels of parasitemia and severe pathogenesis in mice. We found that the simultaneous infection with nonlethal Pb XAT or Py 17X suppressed high levels of parasitemia, liver injury, and body weight loss caused by Pb NK65 infection, induced high levels of reticulocytemia, and subsequently prolonged survival of mice. In coinfected mice, the immune response, including the expansion of B220(int)CD11c(+) cells and CD4(+) T cells and expression of IL-10 mRNA, was comparable to that in nonlethal infection. Moreover, the suppression of liver injury and body weight loss by coinfection was reduced in IL-10(-/-) mice, suggesting that IL-10 plays a role for a reduction of severity by coinfection with nonlethal malaria parasites.     

Motility and infectivity of Plasmodium berghei sporozoites expressing avian Plasmodium gallinaceum circumsporozoite protein

Avian and rodent malaria sporozoites selectively invade different vertebrate cell types, namely macrophages and hepatocytes, and develop in distantly related vector species. To investigate the role of the circumsporozoite (CS) protein in determining parasite survival in different vector species and vertebrate host cell types, we replaced the endogenous CS protein gene of the rodent malaria parasite Plasmodium berghei with that of the avian parasite P. gallinaceum and control rodent parasite P. yoelii. In anopheline mosquitoes, P. berghei parasites carrying P. gallinaceum and rodent parasite P. yoelii CS protein gene developed into oocysts and sporozoites. Plasmodium gallinaceum CS expressing transgenic sporozoites, although motile, failed to invade mosquito salivary glands and to infect mice, which suggests that motility alone is not sufficient for invasion. Notably, a percentage of infected Anopheles stephensi mosquitoes showed melanotic encapsulation of late stage oocysts. This was not observed in control infections or in A. gambiae infections. These findings shed new light on the role of the CS protein in the interaction of the parasite with both the mosquito vector and the rodent host.     

Exacerbation of Plasmodium yoelii malaria in Echinostoma caproni infected mice and abatement through anthelmintic treatment

The effect of chronic intestinal trematode infection on malaria was examined in a murine model of co-infection using Echinostoma caproni and Plasmodium yoelii. BALB/c mice (n = 32) infected with a low dose of E. caproni (approximately 10 cysts) 25-35 days before malaria infection displayed significantly increased malaria parasitemia (P = 0.01), extended patency of malaria (P = 0.03), and increased fatality (47%; P < 0.001) compared to mice infected only with P. yoelii (17X nonlethal strain) (n = 18). Further analysis revealed that differences in malaria parasitemia between fatal co-infections and infections with P. yoelii only were highly significant (P < 0.0001), whereas nonfatal co-infections were not statistically different. Exacerbation of malaria was demonstrated to be reversible through clearance of E. caproni worms by praziquantel treatment administered 10 days before malaria infection. No deaths were observed during malaria infection in mice cleared of their E. caproni infection (n = 10), and parasitemia was significantly reduced from that of untreated co-infected mice (P = 0.03) and was not different from that of mice infected with P. yoelii only. Further studies examining parasite-parasite interactions and host immune response in the echinostome model are warranted to understand the mechanisms affecting the course and outcome of malaria infection during concomitant helminth infection.     

Scant parasitemia in BALB/c mice with congenital malaria infection

Balb/c mice were examined to determine whether or not they transmitted rodent malaria, Plasmodium berghei, to their fetuses. On the 15th day of pregnancy, mice were inoculated with approximately 3 x 10(6) P. berghei-infected erythrocytes by peritoneal injection. The blood from 27 adult females and 196 neonates was examined using a sensitive polymerase chain reaction (PCR) method with a detection level of approximately 1 parasite/microl blood. The average parasitemia of females at delivery was 8.1%, ranging from nondetectable to 37.1%. In 12 females, nested PCR established the presence of blood parasite DNA. Malaria parasites were microscopically confirmed in 2 of the 12 neonates. Maternal parasitemia at the time of delivery was not correlated with the incidence of vertical infection (6.1%), which was higher in this study than that found in previous studies. Although the combination of balb/c mice and P. berghei has not been used to examine vertical transmission of malaria, our report showed that this model may be used for this purpose.     

Deletion of a malaria invasion gene reduces death and anemia, in model hosts

Malaria parasites induce complex cellular and clinical phenotypes, including anemia, cerebral malaria and death in a wide range of mammalian hosts. Host genes and parasite 'toxins' have been implicated in malarial disease, but the contribution of parasite genes remains to be fully defined. Here we assess disease in BALB/c mice and Wistar rats infected by the rodent malaria parasite Plasmodium berghei with a gene knock out for merozoite surface protein (MSP) 7. MSP7 is not essential for infection but in P. falciparum, it enhances erythrocyte invasion by 20%. In vivo, as compared to wild type, the P. berghei Δmsp7 mutant is associated with an abrogation of death and a decrease from 3% to 2% in peak, circulating parasitemia. The Δmsp7 mutant is also associated with less anemia and modest increase in the size of follicles in the spleen. Together these data show that deletion of a single parasite invasion ligand modulates blood stage disease, as measured by death and anemia. This work is the first to assess the contribution of a gene present in all plasmodial species in severe disease.     

Modulation of the Host's Immune Response to Plasmodium berghei by a Parasite-Derived Immunosuppressive Factor

ABSTRACT. It is well known that Plasmodium -infected hosts are immunosuppressed, as shown by their depressed immune responsiveness to a variety of antigens. It is not known, however, whether the immune response of malaria-infected animals to the malarial parasite itself is suppressed. The availability of a noninfectious, immunosuppressive factor (ISF) derived from Plasmodium berghei -infected rat erythrocytes made it possible to investigate this question. Mice infected with P. berghei and injected with the ISF had higher levels of parasitemia and shorter survival times than control mice that were similarly infected but were treated with control material derived from noninfected rat erythrocytes or with saline solution. Conversely, mice immunized against the ISF and then infected with P. berghei had lower parasitemias and longer survival times than mice immunized with the control material or with saline solution. We conclude that immunosuppression in murine malaria affects the course of malaria infection.     

Fas has a role in cerebral malaria, but not in proliferation or exclusion of the murine parasite in mice

We examined the susceptibility of murine Fas-deficient mutants to malaria infection in order to investigate the role of Fas in an experimental murine model of cerebral malaria (CM). We infected mice of B6 and CBA wild-type and mutant backgrounds with Plasmodium berghei ANKA. The incidence of CM in the mutant mice (B6-lpr, CBA-lpr^cg) was decreased by about 50% compared with wild-type control strains at 2 weeks after infection. We did not observe significant differences of parasitemia during a murine malaria infection with nonlethal Plasmodium yoelii 17XNL between wild-type and lymphoproliferative (lpr) mutant mice of C3H and MRL genetic backgrounds, although B6-lpr mice exhibited significantly higher parasitemia than did B6 mice 12 to 18 days after infection. These results suggest Fas has a possible role in CM but may not play a major role in the proliferation or exclusion of a murine malaria parasite in a nonlethal infection.     

Improved methods for haemozoin quantification in tissues yield organ- and parasite-specific information in malariainfected mice

ABSTRACT: BACKGROUND: Despite intensive research, malaria remains a major health concern for non-immune residents and travelers in malaria-endemic regions. Efficient adjunctive therapies against lifethreatening complications such as severe malarial anaemia, encephalopathy, placental malaria or respiratory problems are still lacking. Therefore, new insights into the pathogenesis of severe malaria are imperative. Haemozoin (Hz) or malaria pigment is produced during intraerythrocytic parasite replication, released in the circulation after schizont rupture and accumulates inside multiple organs. Many in vitro and ex vivoimmunomodulating effects are described for Hz but in vivo data are limited. This study aimed to improve methods for Hz quantification in tissues and to investigate the accumulation of Hz in different organs from mice infected with Plasmodium parasites with a varying degree of virulence. METHODS: An improved method for extraction of Hz from tissues was elaborated and coupled to an optimized, quantitative, microtiter plate-based luminescence assay with a high sensitivity. In addition, a technique for measuring Hz by semi-quantitative densitometry, applicable on transmitted light images, was developed. The methods were applied to measure Hz in various organs of C57BL/6J mice infected with Plasmodium berghei ANKA, P. berghei NK65 or Plasmodium chabaudi AS. The used statistical methods were the Mann-Whitney U test and Pearsons correlation analysis. RESULTS: Most Hz was detected in livers and spleens, lower levels in lungs and kidneys, whereas subnanomolar amounts were observed in brains and hearts from infected mice, irrespectively of the parasite strain used. Furthermore, total Hz contents correlated with peripheral parasitaemia and were significantly higher in mice with a lethal P. berghei ANKA or P. berghei NK65-infection than in mice with a self-resolving P. chabaudi AS-infection, despite similar peripheral parasitaemia levels. CONCLUSIONS: The developed techniques were useful to quantify Hz in different organs with a high reproducibility and sensitivity. An organ-specific Hz deposition pattern was found and was independent of the parasite strain used. Highest Hz levels were identified in mice infected with lethal parasite strains suggesting that Hz accumulation in tissues is associated with malaria-related mortality.     

Immunoregulation in murine malaria. Susceptibility of inbred mice to infection with Plasmodium yoelii depends on the dynamic interplay of host and parasite genes

Inbred and H-2 congenic mouse strains were tested for their ability to resist infections with the non-lethal 17X or with the lethal YM isolates of Plasmodium yoelii. DBA/2 and B10.D2 mice, which best resisted infections with non-lethal P. yoelii, were exquisitely susceptible to infection with lethal isolates of this malaria species. In contrast, B6 and B10 mice, which were susceptible to infection with non-lethal P. yoelii, were resistant to infection with the lethal isolates. This reversal of host response phenotype was influenced by H-2 genes, as evidenced by the divergent responses of the H-2 congenic strains B10 and B10.D2. However, a survey of mouse strains sharing common H-2 genes, but expressing different genetic backgrounds, demonstrated that genes outside the H-2 complex also influence the outcome of P. yoelii infections. By enumerating the numbers of P. yoelii-specific antibody-secreting cells in the spleens of infected mice, it was demonstrated that B6 mice, although susceptible to infection with non-lethal P. yoelii, nonetheless made a far stronger anti-parasite response after infection than did resistant DBA/2 mice. Using FACS analysis it was shown that infected B6 mice also produced large amounts of antibodies which bound to the surface of uninfected RBC. Thus, in B6 mice infected with non-lethal P. yoelii, a strong parasite-induced immune response was associated with susceptibility rather than resistance to infection. When T cell-deficient nude mice and their normal littermates were infected with the different isolates of P. yoelii, the nude mice had lower levels of parasitemia and higher RBC counts during the early stages of these infections, and lived longer than did normal littermates after infection with the lethal isolate. These data and the data from studies of B6 and DBA/2 mice support the idea that a strong immune response may be associated with susceptibility rather than resistance to P. yoelii, at least during the early stages of the infection. The finding that a single strain of mouse may present as resistant to infection with one P. yoelii isolate yet be exquisitely susceptible to infection with another suggests that the outcome of these murine malaria infections is dependent on a dynamic interplay between host and parasite genes. Thus, when genetic variability exists in both the host and the parasite populations, as would occur in nature, there may be little directed evolutionary change toward one phenotype or another.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of the host's immune response to Plasmodium berghei by a parasite-derived immunosuppressive factor

It is well known that Plasmodium-infected hosts are immunosuppressed, as show by their depressed immune responsiveness to a variety of antigens. It is not known, however, whether the immune response of malaria-infected animals to the malarial parasite itself is suppressed. The availability of a noninfectious, immunosuppressive factor (ISF) derived from Plasmodium berghei-infected rat erythrocytes made it possible to investigate this question. Mice infected with P. berghei and injected with the ISF had higher levels of parasitemia and shorter survival times than control mice that were similarly infected but were treated with control material derived from noninfected rat erythrocytes or with saline solution. Conversely, mice immunized against the ISF and then infected with P. berghei had lower parasitemias and longer survival times than mice immunized with the control material or with saline solution. We conclude that immunosuppression in murine malaria affects the course of malaria infection.     

The Infection of Duck and Goose Erythrocytes by the Mammalian Malaria Parasite, Plasmodium berghei

The infection of mice and baby rats by both Plasmodium lophurae , an avian parasite, and Plasmodium berghei , a mammalian malaria parasite, prompted investigation of the likelihood of P. berghei infecting avian erythrocytes. Though erythrocytes of chick embryos were not infected, those of the goose and duck embryos were. In both these cells the morphology of the parasite was markedly different from that seen in mammalian erythrocytes. Infections were transitory and it was impossible to find parasites after 4 days. Examination of the hosts of both species of parasites showed a rather wide range and examination of the susceptibility of the duck erythrocyte indicated that this cell was peculiarly receptive to infection by a variety of plasmodia.     

Chromosomal mapping of the host resistance locus to rodent malaria (Plasmodium yoelii) infection in mice

The disease outcome in malaria caused by the protozoan parasite Plasmodium is influenced by host genetic factors. To identify host genes conferring resistance to infection with the malaria parasite, we undertook chromosomal mapping using a whole-genome scanning approach in cross-bred mice. NC/Jic mice all died with high parasitemia within 8 days of infection with 1 x 10(5) parasitized erythrocytes. In contrast, 129/SvJ mice all completely excluded malaria parasites from the circulation and remained alive 21 days after infection. We performed linkage analysis in backcross [(NC/Jic x 129/SvJ)xNC/Jic] mice. The Pymr ( Plasmodium yoelii malaria resistance) locus was mapped to the telomeric portion of mouse Chromosome (Chr) 9. This locus controls host survival and parasitemia after infection. The Char1 locus ( P. chabaudi resistance locus 1), controlling host survival and peak parasitemia in P. chabaudi infection, was previously mapped to the same region. This host resistance locus mapping to Chr 9 may represent a ubiquitous locus controlling susceptibility to rodent malaria. Elucidation of the function of this gene will provide valuable insights into the mechanism of host defense against malaria parasite infection.     

Filaria-induced IL-10 suppresses murine cerebral malaria

Filarial nematodes achieve long survival in their hosts due to their capacity to modulate immune responses. Therefore, immunomodulation by filarial nematodes may alter responses to concomitant infections such as malaria. Cerebral malaria (CM), a severe complication of Plasmodium falciparum infections, is triggered as a consequence of the immune response developed against malaria parasites. The question arises whether prior infection with helminth parasites is beneficial against CM. In the present work a murine model for subsequent has been used to assess this hypothesis. C57BL/6 mice were infected with the rodent filarial parasite Litomosoides sigmodontis and the murine model parasite for CM, Plasmodium berghei ANKA. Previously filaria-infected C57BL/6 mice showed significantly reduced CM rates. CD8⁺ T cell recruitment to the brain, a hallmark for CM development, was reduced in protected mice. Furthermore, in contrast to P. berghei single-infected animals, filaria-infected mice had significantly higher levels of circulating IL-10. The requirement for IL-10 in CM protection was demonstrated by the lack of protection in IL-10 KO mice. This suggests that the anti-inflammatory IL-10 elicited by filarial nematodes is able to suppress the overwhelming inflammatory reaction otherwise triggered against malaria parasites in C57BL/6 mice, preventing full progress to CM.     

Role of IFN-gamma in lethal and nonlethal malaria in susceptible and resistant murine hosts

IFN-gamma plays an important role in host defense against microbial disease. Here, we studied the role of IFN-gamma in lethal and nonlethal murine malaria. Administration of recombinant murine IFN-gamma resulted in a dose-dependent protection of SW, BALB/cByJ, and CBA/J mice from the lethal variant of Plasmodium yoelii 17x (PyL) but had little effect on the course of the nonlethal variant of this parasite (PyNL). Administration of recombinant IFN-gamma also resulted in the activation of peritoneal macrophages for increased phagocytosis of malaria-infected erythrocytes and release of H2O2, as measured in vitro. The ability of spleen cells from infected mice to produce endogenous IFN-gamma and release H2O2 during the course of malaria was also studied. In BALB/cByJ mice, which are relatively susceptible to PyL and PyNL, there was an initial burst of IFN-gamma only in response to PyNL whereas in CBA/J mice, which are relatively resistant to these parasites, there was an initial burst of IFN-gamma in response to both PyL and PyNL. The kinetics of H2O2 release corresponded to that of IFN-gamma. In all infections, levels of IFN-gamma declined as parasitemia increased; however, nonlethal infections were characterized by a recovery of both IFN-gamma activity and H2O2 release as parasitemia declined. These data suggest that IFN-gamma may play an important role in modulating the course of malaria infections by activating macrophages for both intracellular and extracellular parasite destruction.     

Passive immunization against murine malaria with an IgG3 monoclonal antibody

Spleen cells of BALB/c mice that were immune to the 17X strain of P. yoelii were fused with P3X63Ag8 myeloma cells. Two hundred fifty-three of 1053 hybrid cells produced antibodies reactive with disrupted 17X parasites in a solid phase radioimmunoassay. One of these antibodies, McAb 302, reacted with the merozoites of the 17X (nonlethal) and 17XL (lethal) variants of P. yoelii. Of greater significance, McAb 302 passively protected mice against challenge infection with the lethal variant. Mice treated with this antibody before infection developed low-grade parasitemia (less than 0.3%) of short duration when challenged with P. yoelii 17XL . In contrast, control mice that had been untreated or injected with ascites fluid lacking McAb 302 uniformly died with fulminating malaria upon challenge with the same parasite. In other experiments, McAb 302 was shown capable of controlling blood parasite levels when administered to mice with patent P. yoelii 17XL infections. Although all control mice died, mice protected with a single dose of McAb 302 ultimately cleared their infections. Regardless of how passive immunization was performed, mice given McAb 302 were resistant to subsequent challenge with P. yoelii 17XL , indicating they had developed significant immunity during their initial controlled infections. McAb 302 also showed pronounced passive protective activity against the nonlethal 17X strain of P. yoelii, which is a parasite of reticulocytes. The protection afforded by McAb 302 was specific, because mice passively immunized with this antibody died when challenged with the unrelated P. vinckei. McAb 302 was shown to possess the IgG3 isotype and precipitated a 230-kd protein plus several smaller polypeptides from metabolically labeled parasite antigen preparation derived from both variants of P. yoelii. It did not react with similar preparations of other murine plasmodial species.