A fast and cost-effective microsampling protocol incorporating reduced animal usage for time-series transcriptomics in rodent malaria parasites.

BACKGROUND: The transcriptional regulation that occurs in malaria parasites during the erythrocytic stages of infection can be studied in vivo with rodent malaria parasites propagated in mice. Time-series transcriptome profiling commonly involves the euthanasia of groups of mice at specific time points followed by the extraction of parasite RNA from whole blood samples. Current methodologies for parasite RNA extraction involve several steps and when multiple time points are profiled, these protocols are laborious, time-consuming, and require the euthanization of large cohorts of mice. RESULTS: A simplified protocol has been designed for parasite RNA extraction from blood volumes as low as 20 µL (microsamples), serially bled from mice via tail snips and directly lysed with TRIzol reagent. Gene expression data derived from microsampling using RNA-seq were closely matched to those derived from larger volumes of leucocyte-depleted and saponin-treated blood obtained from euthanized mice with high reproducibility between biological replicates. Transcriptome profiling of microsamples taken at different time points during the intra-erythrocytic developmental cycle of the rodent malaria parasite Plasmodium vinckei revealed the transcriptional cascade commonly observed in malaria parasites. CONCLUSIONS: Microsampling is a quick, robust and cost-efficient approach to sample collection for in vivo time-series transcriptomic studies in rodent malaria parasites.

A Novel Model of Asymptomatic Plasmodium Parasitemia That Recapitulates Elements of the Human Immune Response to Chronic Infection.

In humans, immunity to Plasmodium sp. generally takes the form of protection from symptomatic malaria (i.e., 'clinical immunity') rather than infection ('sterilizing immunity'). In contrast, mice infected with Plasmodium develop sterilizing immunity, hindering progress in understanding the mechanistic basis of clinical immunity. Here we present a novel model in which mice persistently infected with P. chabaudi exhibit limited clinical symptoms despite sustaining patent parasite burdens for many months. Characterization of immune responses in persistently infected mice revealed development of CD4+ T cell exhaustion, increased production of IL-10, and expansion of B cells with an atypical surface phenotype. Additionally, persistently infected mice displayed a dramatic increase in circulating nonclassical monocytes, a phenomenon that we also observed in humans with both chronic Plasmodium exposure and asymptomatic infection. Following pharmacological clearance of infection, previously persistently infected mice could not control a secondary challenge, indicating that persistent infection disrupts the sterilizing immunity that typically develops in mouse models of acute infection. This study establishes an animal model of asymptomatic, persistent Plasmodium infection that recapitulates several central aspects of the immune response in chronically exposed humans. As such, it provides a novel tool for dissection of immune responses that may prevent development of sterilizing immunity and limit pathology during infection.

Heterologous Infection of Pregnant Mice Induces Low Birth Weight and Modifies Offspring Susceptibility to Malaria.

Pregnancy malaria (PM) is associated with poor pregnancy outcomes, and can arise due to relapse, recrudescence or a re-infection with heterologous parasites. We have used the Plasmodium chabaudi model of pregnancy malaria in C57BL/6 mice to examine recrudescence and heterologous infection using CB and AS parasite strains. After an initial course of patent parasitemia and first recrudescence, CB but not AS parasites were observed to recrudesce again in most animals that became pregnant. Pregnancy exacerbated heterologous CB infection of AS-experienced mice, leading to mortality and impaired post-natal growth of pups. Parasites were detected in placental blood without evidence of sequestration, unlike P. falciparum but similar to other malaria species that infect pregnant women. Inflammatory cytokine levels were elevated in pregnant females during malaria, and associated with intensity of infection and with poor outcomes. Pups born to dams during heterologous infection were more resistant to malaria infections at 6-7 weeks of age, compared to pups born to malaria-experienced but uninfected dams or to malaria-naïve dams. In summary, our mouse model reproduces several features of human PM, including recrudescences, heterologous infections, poor pregnancy outcomes associated with inflammatory cytokines, and modulation of offspring susceptibility to malaria. This model should be further studied to explore mechanisms underlying PM pathogenesis.

Infection with Plasmodium chabaudi diminishes plasma immune complexes and ameliorates the histopathological alterations in different organs of female BWF1 lupus mice.

OBJECTIVE: To determine the effect of Plasmodium chabaudi infection on the plasma level of circulating immune complexes (CICs), haemoglobin (Hb) content, urine profile, and histological features of female BWF1 mice, the murine model of systemic lupus erythematosus (SLE). MATERIALS AND METHODS: A total of 30 female BWF1 lupus mice were randomly divided into three groups as follows: group (I) control group (P. chabaudi uninfected); group (II) lupus mice infected with live P. chabaudi; group (III) lupus mice infected with irradiated P. chabaudi. Urine samples were daily collected from the second week-post infection. Mice from the three groups were killed at day 14 post-infection and heparinized blood was collected for further haemoglobin contents and plasma analysis. Paraffin-embedded kidney, liver, lung, heart, brain, ovary and skin tissues were stained with Hematoxylin and Eosin (H&E) and examined under light microscope. RESULTS: Our results reveal that infection of lupus mice with live P. chabaudi was associated with an increase in urinary Hb and a decrease in plasma Hb and CIC levels. Interestingly, infection of lupus mice with live P. chabaudi ameliorates the histopathological alterations mediated by lupus disease in kidney tissues. Although no parasite sequestration was observed in any of the investigated organs, P. chabaudi pigment deposition was observed in the liver of both live and irradiated P. chabaudi infected groups. CONCLUSIONS: This study in lupus prone BWF1 mice indicated that gamma-irradiated P. chabaudi infection has the desired lupus ameliorating effect without negative effects of malaria which assist the understanding of different responses to plasmodium sp. infection in human lupus patients.

The antiplasmodial and spleen protective role of crude Indigofera oblongifolia leaf extract traditionally used in the treatment of malaria in Saudi Arabia.

Malaria is one of the most serious natural hazards faced by human society. Although plant leaves of Indigofera oblongifolia have been used for the treatment of malaria in Saudi Arabian society, there is no laboratory-based evidence for the effectiveness and safety of the plant. This study therefore was designed to investigate the antimalarial and spleen protective activity of I. oblongifolia leaf extract (IOLE) in mice. Three doses (100, 200 and 300 mg/kg) of IOLE were used to treat mice infected with Plasmodium chabaudi-parasitized erythrocytes. The suppressive effect produced by the 100 mg/kg dose on parasitemia was highly significant compared to the infected nontreated group. This dose was also able to repair the change in the thickness of the mice spleen and significantly lower the number of apoptotic cells in the spleen. Moreover, I. oblongifolia also altered gene expression in the infected spleen. On day 7 postinfection, the mRNA expression of six genes - with immune response functions - was upregulated by more than twofold, while that of 24 other genes was downregulated. Among the differentially up- and downregulated genes under the effect of IOLE, we quantified the expression of Ccl8, Saa3, Cd209a, and Cd209b mRNAs. The expression data, determined by microarrays, were largely consistent with the expression analyses we performed with several arbitrarily selected genes using quantitative polymerase chain reaction (PCR). Based on our results, I. oblongifolia exhibits antimalarial activity and could protect the spleen from P. chabaudi-induced injury.

In vivo assessment of rodent Plasmodium parasitemia and merozoite invasion by flow cytometry.

During blood stage infection, malaria parasites invade, mature, and replicate within red blood cells (RBCs). This results in a regular growth cycle and an exponential increase in the proportion of malaria infected RBCs, known as parasitemia. We describe a flow cytometry based protocol which utilizes a combination of the DNA dye Hoechst, and the mitochondrial membrane potential dye, JC-1, to identify RBCs which contain parasites and therefore the parasitemia, of in vivo blood samples from Plasmodium chabaudi adami DS infected mice. Using this approach, in combination with fluorescently conjugated antibodies, parasitized RBCs can be distinguished from leukocytes, RBC progenitors, and RBCs containing Howell-Jolly bodies (HJ-RBCs), with a limit of detection of 0.007% parasitemia. Additionally, we outline a method for the comparative assessment of merozoite invasion into two different RBC populations. In this assay RBCs, labeled with two distinct compounds identifiable by flow cytometry, are transfused into infected mice. The relative rate of invasion into the two populations can then be assessed by flow cytometry based on the proportion of parasitized RBCs in each population over time. This combined approach allows the accurate measurement of both parasitemia and merozoite invasion in an in vivo model of malaria infection.

Co-infection restrains Litomosoides sigmodontis filarial load and plasmodial P. yoelii but not P. chabaudi parasitaemia in mice.

Infection with multiple parasite species is clearly the norm rather than the exception, in animals as well as in humans. Filarial nematodes and Plasmodium spp. are important parasites in human public health and they are often co-endemic. Interactions between these parasites are complex. The mechanisms underlying the modulation of both the course of malaria and the outcome of filarial infection are poorly understood. Despite increasing activity in recent years, studies comparing co- and mono-infections are very much in their infancy and results are contradictory at first sight. In this study we performed controlled and simultaneous co-infections of BALB/c mice with Litomosoides sigmodontis filaria and with Plasmodium spp. (Plasmodium yoelii 17 XNL or Plasmodium chabaudi 864VD). An analysis of pathological lesions in the kidneys and lungs and a parasitological study were conducted at different times of infection. Whatever the plasmodial species, the filarial recovery rate was strongly decreased. The peak of parasitaemia in the plasmodial infection was decreased in the course of P. yoelii infection but not in that of P. chabaudi. Regarding pathological lesions, L. sigmodontis can reverse lesions in the kidneys due to the presence of both Plasmodium species but does not modify the course of pulmonary lesions. The filarial infection induces granulomas in the lungs.

A flow cytometric assay to quantify invasion of red blood cells by rodent Plasmodium parasites in vivo.

BACKGROUND: Malaria treatments are becoming less effective due to the rapid spread of drug resistant parasites. Increased understanding of the host/parasite interaction is crucial in order to develop treatments that will be less prone to resistance. Parasite invasion of the red blood cell (RBC) is a critical aspect of the parasite life cycle and is, therefore, a promising target for the development of malaria treatments. Assays for analysing parasite invasion in vitro have been developed, but no equivalent assays exist for in vivo studies. This article describes a novel flow cytometric in vivo parasite invasion assay. METHODS: Experiments were conducted with mice infected with erythrocytic stages of Plasmodium chabaudi adami strain DS. Exogenously labelled blood cells were transfused into infected mice at schizogony, and collected blood samples stained and analysed using flow cytometry to specifically detect and measure proportions of labelled RBC containing newly invaded parasites. A combination of antibodies (CD45 and CD71) and fluorescent dyes, Hoechst (DNA) and JC-1 (mitochondrial membrane potential), were used to differentiate parasitized RBCs from uninfected cells, RBCs containing Howell-Jolly bodies, leukocytes and RBC progenitors. Blood cells were treated ex vivo with proteases to examine the effects on in vivo parasite invasion. RESULTS: The staining and flow cytometry analysis method was accurate in determining the parasitaemia down to 0.013% with the limit of detection at 0.007%. Transfused labelled blood supported normal rates of parasite invasion. Protease-treated red cells resulted in 35% decrease in the rate of parasite invasion within 30 minutes of introduction into the bloodstream of infected mice. CONCLUSIONS: The invasion assay presented here is a versatile method for the study of in vivo red cell invasion efficiency of Plasmodium parasites in mice, and allows direct comparison of invasion in red cells derived from two different populations. The method also serves as an accurate alternative method of estimating blood parasitaemia.

Vector transmission regulates immune control of Plasmodium virulence.

Defining mechanisms by which Plasmodium virulence is regulated is central to understanding the pathogenesis of human malaria. Serial blood passage of Plasmodium through rodents, primates or humans increases parasite virulence, suggesting that vector transmission regulates Plasmodium virulence within the mammalian host. In agreement, disease severity can be modified by vector transmission, which is assumed to 'reset' Plasmodium to its original character. However, direct evidence that vector transmission regulates Plasmodium virulence is lacking. Here we use mosquito transmission of serially blood passaged (SBP) Plasmodium chabaudi chabaudi to interrogate regulation of parasite virulence. Analysis of SBP P. c. chabaudi before and after mosquito transmission demonstrates that vector transmission intrinsically modifies the asexual blood-stage parasite, which in turn modifies the elicited mammalian immune response, which in turn attenuates parasite growth and associated pathology. Attenuated parasite virulence associates with modified expression of the pir multi-gene family. Vector transmission of Plasmodium therefore regulates gene expression of probable variant antigens in the erythrocytic cycle, modifies the elicited mammalian immune response, and thus regulates parasite virulence. These results place the mosquito at the centre of our efforts to dissect mechanisms of protective immunity to malaria for the development of an effective vaccine.

Artemisinin resistance in rodent malaria--mutation in the AP2 adaptor µ-chain suggests involvement of endocytosis and membrane protein trafficking.

BACKGROUND: The control of malaria, caused by Plasmodium falciparum, is hampered by the relentless evolution of drug resistance. Because artemisinin derivatives are now used in the most effective anti-malarial therapy, resistance to artemisinin would be catastrophic. Indeed, studies suggest that artemisinin resistance has already appeared in natural infections. Understanding the mechanisms of resistance would help to prolong the effective lifetime of these drugs. Genetic markers of resistance are therefore required urgently. Previously, a mutation in a de-ubiquitinating enzyme was shown to confer artemisinin resistance in the rodent malaria parasite Plasmodium chabaudi. METHODS: Here, for a mutant P. chabaudi malaria parasite and its immediate progenitor, the in vivo artemisinin resistance phenotypes and the mutations arising using Illumina whole-genome re-sequencing were compared. RESULTS: An increased artemisinin resistance phenotype is accompanied by one non-synonymous substitution. The mutated gene encodes the µ-chain of the AP2 adaptor complex, a component of the endocytic machinery. Homology models indicate that the mutated residue interacts with a cargo recognition sequence. In natural infections of the human malaria parasite P. falciparum, 12 polymorphisms (nine SNPs and three indels) were identified in the orthologous gene. CONCLUSION: An increased artemisinin-resistant phenotype occurs along with a mutation in a functional element of the AP2 adaptor protein complex. This suggests that endocytosis and trafficking of membrane proteins may be involved, generating new insights into possible mechanisms of resistance. The genotypes of this adaptor protein can be evaluated for its role in artemisinin responses in human infections of P. falciparum.

Preconditioning with hemin decreases Plasmodium chabaudi adami parasitemia and inhibits erythropoiesis in BALB/c mice.

Increased susceptibility to bacterial and viral infections and dysfunctional erythropoiesis are characteristic of malaria and other hemolytic hemoglobinopathies. High concentrations of free heme are common in these conditions but little is known about the effect of heme on adaptive immunity and erythropoiesis. Herein, we investigated the impact of heme (hemin) administration on immune parameters and steady state erythropoiesis in BALB/c mice, and on parasitemia and anemia during Plasmodium chabaudi adami infection. Intra-peritoneal injection of hemin (5 mg/Kg body weight) over three consecutive days decreased the numbers of splenic and bone marrow macrophages, IFN-γ responses to CD3 stimulation and T(h)1 differentiation. Our results show that the numbers of erythroid progenitors decreased in the bone marrow and spleen of mice treated with hemin, which correlated with reduced numbers of circulating reticulocytes, without affecting hemoglobin concentrations. Although blunted IFN-γ responses were measured in hemin-preconditioned mice, the mice developed lower parasitemia following P.c.adami infection. Importantly, anemia was exacerbated in hemin-preconditioned mice with malaria despite the reduced parasitemia. Altogether, our data indicate that free heme has dual effects on malaria pathology.

Mosquito transmission of the rodent malaria parasite Plasmodium chabaudi.

BACKGROUND: Serial blood passage of Plasmodium increases virulence, whilst mosquito transmission inherently regulates parasite virulence within the mammalian host. It is, therefore, imperative that all aspects of experimental malaria research are studied in the context of the complete Plasmodium life cycle. METHODS: Plasmodium chabaudi chabaudi displays many characteristics associated with human Plasmodium infection of natural mosquito vectors and the mammalian host, and thus provides a unique opportunity to study the pathogenesis of malaria in a single infection setting. An optimized protocol that permits efficient and reproducible vector transmission of P. c. chabaudi via Anopheles stephensi was developed. RESULTS AND CONCLUSIONS: This protocol was utilized for mosquito transmission of genetically distinct P. c. chabaudi isolates, highlighting differential parasite virulence within the mosquito vector and the spectrum of host susceptibility to infection initiated via the natural route, mosquito bite. An apposite experimental system in which to delineate the pathogenesis of malaria is described in detail.

Direct loop-mediated isothermal amplification from Plasmodium chabaudi infected blood samples: inability to discriminate genomic and cDNA sequences.

Loop-mediated isothermal amplification (LAMP) has been increasingly used for diagnosis and quantification of pathogens. Since the Bst DNA polymerase used in this assay is highly resistant to PCR inhibitors present in blood, direct analysis of blood samples without DNA or RNA extraction is possible. Indeed, the presence of Plasmodium chabaudi specific nucleic acids was easily detectable using primer sets for P. chabaudi 18S rRNA and the cir 1 mRNA. Despite the fact that primers for cir 1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and actin II mRNAs were used that spanned an intron, selective amplification of mRNA in the presence of contaminating genomic DNA was not possible. Optimization of the reaction temperature could only improve discrimination when low complexity templates (target sequences cloned in a plasmid vector) were used. Placing different LAMP primers across intron exon boundaries did not prevent amplification in the absence of reverse transcriptase. Probably due to the high A+T content and low number of introns only a very limited number of possible primer sets spanning introns could be identified in the target genes and no reaction conditions could be established that would allow quantification of RNA levels in the presence of DNA directly from blood samples.

Plasmodium chabaudi chabaudi malaria parasites can develop stable resistance to atovaquone with a mutation in the cytochrome b gene.

BACKGROUND: Plasmodium falciparum, has developed resistance to many of the drugs in use. The recommended treatment policy is now to use drug combinations. The atovaquone-proguanil (AP) drug combination, is one of the treatment and prophylaxis options. Atovaquone (ATQ) exerts its action by inhibiting plasmodial mitochondria electron transport at the level of the cytochrome bc1 complex. Plasmodium falciparum in vitro resistance to ATQ has been associated with specific point mutations in the region spanning codons 271-284 of the cytochrome b gene. ATQ -resistant Plasmodium yoelii and Plasmodium berghei lines have been obtained and resistant lines have amino acid mutations in their CYT b protein sequences. Plasmodium chabaudi model for studying drug-responses and drug-resistance selection is a very useful rodent malaria model but no ATQ resistant parasites have been reported so far. The aim of this study was to determine the ATQ sensitivity of the P. chabaudi clones, to select a resistant parasite line and to perform genotypic characterization of the cytb gene of these clones. METHODS: To select for ATQ resistance, Plasmodium. chabaudi chabaudi clones were exposed to gradually increasing concentrations of ATQ during several consecutive passages in mice. Plasmodium chabaudi cytb gene was amplified and sequenced. RESULTS: ATQ resistance was selected from the clone AS-3CQ. In order to confirm whether an heritable genetic mutation underlies the response of AS-ATQ to ATQ, the stability of the drug resistance phenotype in this clone was evaluated by measuring drug responses after (i) multiple blood passages in the absence of the drug, (ii) freeze/thawing of parasites in liquid nitrogen and (iii) transmission through a mosquito host, Anopheles stephensi. ATQ resistance phenotype of the drug-selected parasite clone kept unaltered. Therefore, ATQ resistance in clone AS-ATQ is genetically encoded. The Minimum Curative Dose of AS-ATQ showed a six-fold increase in MCD to ATQ relative to AS-3CQ. CONCLUSIONS: A mutation was found on the P. chabaudi cytb gene from the AS-ATQ sample a substitution at the residue Tyr268 for an Asn, this mutation is homologous to the one found in P. falciparum isolates resistant to ATQ.

The severity of malarial anaemia in Plasmodium chabaudi infections of BALB/c mice is determined independently of the number of circulating parasites.

BACKGROUND: Severe malarial anaemia is a major complication of malaria infection and is multi-factorial resulting from loss of circulating red blood cells (RBCs) from parasite replication, as well as immune-mediated mechanisms. An understanding of the causes of severe malarial anaemia is necessary to develop and implement new therapeutic strategies to tackle this syndrome of malaria infection. METHODS: Using analysis of variance, this work investigated whether parasite-destruction of RBCs always accounts for the severity of malarial anaemia during infections of the rodent malaria model Plasmodium chabaudi in mice of a BALB/c background. Differences in anaemia between two different clones of P. chabaudi were also examined. RESULTS: Circulating parasite numbers were not correlated with the severity of anaemia in either BALB/c mice or under more severe conditions of anaemia in BALB/c RAG2 deficient mice (lacking T and B cells). Mice infected with P. chabaudi clone CB suffered more severe anaemia than mice infected with clone AS, but this was not correlated with the number of parasites in the circulation. Instead, the peak percentage of parasitized RBCs was higher in CB-infected animals than in AS-infected animals, and was correlated with the severity of anaemia, suggesting that the availability of uninfected RBCs was impaired in CB-infected animals. CONCLUSION: This work shows that parasite numbers are a more relevant measure of parasite levels in P. chabaudi infection than % parasitaemia, a measure that does not take anaemia into account. The lack of correlation between parasite numbers and the drop in circulating RBCs in this experimental model of malaria support a role for the host response in the impairment or destruction of uninfected RBC in P. chabaudi infections, and thus development of acute anaemia in this malaria model.

Lactobacillus casei ssp. rhamnosus enhances non specific protection against Plasmodium chabaudi AS in mice

OBJECTIVE: To evaluate the capacity of Lactobacillus casei ssp. rhamnosus to enhance resistance against Plasmodium chabaudi chabaudi AS. MATERIAL AND METHODS: NIH mice were IP injected with viable lactobacillus casei seven days (LC1 group) or 7 and 14 days (LC2 group) before the challenge (day 0) with Plasmodium chabaudi parasitized red blood cells (pRBC). Control mice were inoculated with pRBC only. When parasitaemia was resolved, naive mice were injected with spleen cells from each group. The parasitaemia was measured. Nitric oxide (NO.) in serum was determined. RESULTS: Mice from the LC1 group presented a reduction in parasitaemia, with a prepatent period of five days, parasitaemia lasted 11 days, and the peak was (36.3 percent pRBC) on the 12th day post-infection. Mice from the LC2 group showed a prepatent period of five days, parasitaemia lasted eight days, and the peak (30 percent pRBC) was of on the 11th day. In the control, the prepatent period was three days, the parasitaemia lasted 15 days, and the peak (51 percent pRBC) was on day nine. Mice inoculated with spleen cells from the LC2 group showed a prepatent period of 21 days, parasitaemia lasted seven days, and the peak (13.5 percent pRBC) was on the 26th day. CONCLUSION: L. casei enhanced nonspecific resistance to P. chabaudi, as indicated by longer prepatent periods, reduced parasitaemia, and reduction in the viability of the parasites recovered from the spleen of infected mice, along with high concentrations of NO. in serum.

OBJETIVO: Evaluar la capacidad de Lactobacillus casei de aumentar la resistencia a la infección con Plasmodium chabaudi en ratones. MATERIAL Y MÉTODOS: Ratones NIH fueron inyectados intraperitonealmente con L. casei viable 7 días (grupo LC1) o 7 y 14 días (grupo LC2) antes del reto (día 0) con glóbulos rojos parasitados (GRP) con P. chabaudi. Los testigos fueron inoculados con GRP solamente. Cuando la parasitemia se resolvió, se inocularon ratones limpios con células de bazo de cada grupo. Se midió la concentración de óxido nítrico (NO.) en suero. RESULTADOS: El grupo LC1 presentó un periodo prepatente de 5 días, una parasitemia de 11 días con el máximo (36.3 por ciento de GRP) el día 12. Los ratones del grupo LC2 mostraron un periodo prepatente de 5 días, una parasitemia de 8 días con el pico (30 por ciento de GRI) el día 11. En los testigos el periodo prepatente fue de 3 días, la parasitemia de 15 y su máximo (51 por ciento de GRI) el día 9. Los ratones que recibieron células de bazo del grupo LC2, mostraron un período prepatente de 21 días, una parasitemia de 7 con su máximo (13.5 por ciento de GRI) el día 26. CONCLUSION: L. casei aumenta la resistencia no específica hacia P. chabaudi a juzgar por los periodos prepatentes más largos, las bajas parasitemias, la reducción en la viabilidad y la elevación de la concentración de NO. en el suero, que presentaron los ratones estimulados con lactobacilos.

Plasmodium chabaudi: reverse transcription PCR for the detection and quantification of transmission stage malaria parasites.

We have developed two reverse transcription polymerase chain reaction (RT-PCR) techniques to detect and quantify the transmission stages (gametocytes) of Plasmodium chabaudi malaria parasites. Both the qualitative and quantitative techniques are based on the amplification of mRNA coding for the P. chabaudi protein Pcs230, which is expressed exclusively in gametocytes. The quantitative RT-PCR (qRT-PCR) technique was developed and validated by examining serial dilutions of known gametocyte densities. The method generated a high correlation between calibration curves of blind samples (R(2)=0.86). The technique was found to be specific, reproducible, and time efficient for quantification of both patent and sub-patent gametocytemia with a sensitivity level 100-1000 times greater than microscopy. The qualitative RT-PCR (RT-PCR) technique was used to monitor the persistence and dynamics of P. chabaudi gametocytes following acute infection. Mice in two independent experiments were sampled for up to 87 days post-infection. RT-PCR showed that gametocytes can persist for up to 8 weeks, post-infection, whereas microscopy could only detect gametocytes up to 6 weeks. Potential applications of the above techniques for studying the ecology, evolution, and epidemiology of malaria transmission are discussed.

Lactobaciilus casei ssp. rhamnosus enhances non specific protection against Plasmodium chabaudi AS in mice.

OBJECTIVE: To evaluate the capacity of Lactobacillus casei ssp. rhamnosus to enhance resistance against Plasmodium chabaudi chabaudi AS. MATERIAL AND METHODS: NIH mice were IP injected with viable lactobacillus casei seven days (LC1 group) or 7 and 14 days (LC2 group) before the challenge (day 0) with Plasmodium chabaudi parasitized red blood cells (pRBC). Control mice were inoculated with pRBC only. When parasitaemia was resolved, naive mice were injected with spleen cells from each group. The parasitaemia was measured. Nitric oxide (NO*) in serum was determined. RESULTS: Mice from the LC1 group presented a reduction in parasitaemia, with a prepatent period of five days, parasitaemia lasted 11 days, and the peak was (36.3 % pRBC) on the 12th day post-infection. Mice from the LC2 group showed a prepatent period of five days, parasitaemia lasted eight days, and the peak (30 % pRBC) was of on the 11th day. In the control, the prepatent period was three days, the parasitaemia lasted 15 days, and the peak (51% pRBC) was on day nine. Mice inoculated with spleen cells from the LC2 group showed a prepatent period of 21 days, parasitaemia lasted seven days, and the peak (13.5% pRBC) was on the 26th day. CONCLUSION: L. casei enhanced nonspecific resistance to P. chabaudi, as indicated by longer prepatent periods, reduced parasitaemia, and reduction in the viability of the parasites recovered from the spleen of infected mice, along with high concentrations of NO* in serum.

An AFLP-based genetic linkage map of Plasmodium chabaudi chabaudi.

BACKGROUND: Plasmodium chabaudi chabaudi can be considered as a rodent model of human malaria parasites in the genetic analysis of important characters such as drug resistance and immunity. Despite the availability of some genome sequence data, an extensive genetic linkage map is needed for mapping the genes involved in certain traits. METHODS: The inheritance of 672 Amplified Fragment Length Polymorphism (AFLP) markers from two parental clones (AS and AJ) of P. c. chabaudi was determined in 28 independent recombinant progeny clones. These, AFLP markers and 42 previously mapped Restriction Fragment Length Polymorphism (RFLP) markers (used as chromosomal anchors) were organized into linkage groups using Map Manager software. RESULTS: 614 AFLP markers formed linkage groups assigned to 10 of 14 chromosomes, and 12 other linkage groups not assigned to known chromosomes. The genetic length of the genome was estimated to be about 1676 centiMorgans (cM). The mean map unit size was estimated to be 13.7 kb/cM. This was slightly less then previous estimates for the human malaria parasite, Plasmodium falciparum CONCLUSION: The P. c. chabaudi genetic linkage map presented here is the most extensive and highly resolved so far available for this species. It can be used in conjunction with the genome databases of P. c chabaudi, P. falciparum and Plasmodium yoelii to identify genes underlying important phenotypes such as drug resistance and strain-specific immunity.

Amplified fragment length polymorphism measures proportions of malaria parasites carrying specific alleles in complex genetic mixtures.

We are interested in developing a method for the identification of those genes in malaria parasites which underlie a variety of selectable phenotypes of the parasites including drug resistance and strain-specific immunity. A key aspect of our approach is to subject a genetically mixed population of malaria parasites to a specific phenotypic selection pressure such as the administration of an antimalarial drug and then identify genetic markers affected by the selection. Our aim, therefore, is to be able to identify those genetic markers carried by sensitive parasites which disappear from the population after selection as they should be closely linked to the locus determining the phenotype involved. We have previously identified more than 800 amplified fragment length polymorphisms (AFLP) distinguishing two cloned strains of the rodent malaria parasite Plasmodium chabaudi chabaudi and distributed across the whole of the parasites' genome. Here we evaluate the possibility that the intensities of these AFLP bands are quantitatively related to the proportions of parasite DNA which bear these markers in mixtures of genetically different parasites. We prepared mixtures of DNA and parasitised blood from different mixtures of two genetically distinct clones (AS and AJ) of P. c. chabaudi and analysed AFLP markers amplified from them. The results show that the relative band intensities of AFLP markers are, indeed, linearly related to the proportions of parasite DNA in a genetically mixed sample. The precision of the method is sufficient to detect reliably the effects of phenotypic selection at loci closely linked to a genetic locus under selection.