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Following the bite of an infective mosquito, malaria parasites first invade the liver where they develop and replicate for a number of days before being released into the bloodstream where they invade red blood cells and cause disease. The biology of the liver stages of malaria parasites is relatively poorly understood due to the inaccessibility of the parasites to sampling during this phase of their life cycle. Here we report the detection in blood and faecal samples of malaria parasite DNA throughout their development in the livers of mice and before the parasites begin their growth in the blood circulation. It is shown that parasite DNA derived from pre-erythrocytic stage parasites reaches the faeces via the bile. We then show that different primate malaria species can be detected by PCR in blood and faecal samples from naturally infected captive macaque monkeys. These results demonstrate that pre-erythrocytic parasites can be detected and quantified in experimentally infected animals. Furthermore, these results have important implications for both molecular epidemiology and phylogenetics of malaria parasites. In the former case, individuals who are malaria parasite negative by microscopy, but PCR positive for parasite DNA in their blood, are considered to be "sub-microscopic" blood stage parasite carriers. We now propose that PCR positivity is not necessarily an indicator of the presence of blood stage parasites, as the DNA could derive from pre-erythrocytic parasites. Similarly, in the case of molecular phylogenetics based on DNA sequences alone, we argue that DNA amplified from blood or faeces does not necessarily come from a parasite species that infects the red blood cells of that particular host.

Plasmodium falciparum SURFIN 4.2 (PFD1160w) is a polymorphic protein expressed on the surface of parasite-infected erythrocytes. Such molecules are expected to be under strong host immune pressure, thus we analyzed the nucleotide diversity of the N-terminal extracellular region of SURFIN 4.2 using P. falciparum isolates obtained from a malaria hypoendemic area of Thailand. The extracellular region of SURFIN 4.2 was divided into four regions based on the amino acid sequence conservation among SURFIN members and the level of polymorphism among SURFIN 4.2 sequences; N-terminal segment (Nter), a cysteine-rich domain (CRD), a variable region 1 (Var1), and a variable region 2 (Var2). Comparison between synonymous and non-synonymous substitutions, Tajima's D test, and Fu and Li's D* and F* tests detected signatures of positive selection on Var2 and to a lesser extent Var1, suggesting that these regions were likely under host immune pressure. Strong linkage disequilibrium was detected for nucleotide pairs separated by a distance of more than 1.5 kb, and 7 alleles among 19 alleles detected in 1988-1989 still circulated 14 years later, suggesting low recombination of the analyzed surf 4.2 sequence region in Thailand. The allele frequency distribution of polymorphic areas in Var2 did not differ between two groups collected in different time points, suggesting the allele frequency distribution of this region was stable for 14 years. The observed allele frequency distribution of SURFIN 4.2 Var2 may be fixed in Thai P. falciparum population as similar to the observation for P. falciparum merozoite surface protein 1, for which a stable allele frequency distribution was reported., Parasitology International, 61(2), pp.317-323; 2012

Understanding why some malaria-infected individuals are infective to mosquitoes while others are not, is of great importance when considering interventions to stop malaria transmission. Whether gametocytes are produced in every individual infected with Plasmodium falciparum remains unclear. Using a highly sensitive reverse transcription (RT)-PCR assay, we attempted to detect gametocyte-specific mRNA transcripts in isolates from Thai patients which newly adapted to continuous in vitro culture. We then compared the allelic types of the pfg377 gene between patient blood and culture-adapted parasites in order to determine whether the same parasite lines were producing gametocytes in vivo and in vitro. Transcripts of pfg377 were detected in all parasite isolates and in the corresponding cultured isolates, revealing that all patients had gametocytes circulating in their blood at the time of sampling. For isolates in continuous in vitro culture, there was a match between pfg377 allelic types detected by PCR from genomic DNA (and thus indicative of the dominant allelic type of asexual parasites) and those detected by RT-PCR of mRNA (gametocyte-specific), whereas in freshly isolated patient blood there were some differences between the asexual parasite allelic type and that of the gametocytes in the same infection. Seven isolates contained asexual stage parasites harbouring pfg377 alleles that were not detectable in gametocytes from the same infections, suggesting that some clones were not producing gametocytes at the time of sampling, or that they were below the level of detection., International Journal for Parasitology, 41(3-4), pp.317-323; 2011

The feasibility of identifying parasite DNA and specific mRNAs from wild-caught Anopheles dirus mosquitoes was assessed using dried mosquito salivary glands preserved on filter paper. We were able to detect Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium knowlesi DNA by conventional PCR and, furthermore, detected P. falciparum gametocyte-specific genes, pfg377 and pfs16 mRNA, P. knowlesi circumsporozoite protein (CSP) and sporozoite surface protein 2 (SSP2) mRNA by reverse transcription-PCR. Using this technique, we were able to confirm the presence of P. vivax, P. falciparum and P. knowlesi in one particular wild-caught mosquito. These results indicate that P. knowlesi may be transmitted by the primary human malaria vector in forested areas in Vietnam. This study also shows that the preservation of mosquito salivary glands on filter paper, and the down-stream extraction of parasite DNA and RNA from those, offers a powerful resource for molecular epidemiological studies on malaria., International journal for parasitology, 39(14), pp.1533-1537; 2009

We examined the utility of dried blood on filter paper for the source of cytokine messenger RNA (mRNA). Total RNA was isolated from the dried blood of mice infected with Plasmodium yoelii, and cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR). As a reference, we extracted total RNA from peripheral blood collected at the same time as the preparation for dried blood. There was no difference in cytokine mRNA expression between the two sources; the dried blood on filter paper and the peripheral blood. Th1 cells, Th2 cells, and monocytes/macrophages derived cytokine mRNAs in the dried blood from infected mice were detected, and the increase of some of the cytokines mRNAs after infection was also observed. These results suggested that the dried blood on filter paper is satisfactory RNA source for immunological examination in field-based studies.

CD4(+) T cells co-expressing CD25 (CD4(+)CD25(+) T cells) have been identified as immunoregulatory suppressors modulating autoimmune response. Beside that, autoimmune response was supposed to be associated with malaria infection. Based on these data, we hypothesised that CD4(+)CD25(+) T cells may influence protective immunity to malaria parasites, while suppressing autoimmune response arising throughout the course of malarial infection. To test this possibility, we evaluated the kinetics of CD4(+)CD25(+) T cells during malaria infection and investigated the influence of CD25 depletion by anti-mouse CD25 monoclonal antibody (PC61) on the infection, using a mouse model of premunition to Plasmodium berghei NK65 malaria. The results showed that, during exacerbation of P. berghei NK65 infection, the proportion of CD4(+)CD25(+) T cells among CD4(+) T cells decreased, although that of CD4(+) T cells increased. CD25 depletion clearly delayed the growth of parasitaemia during parasite challenge, particularly in immunised mice. These findings demonstrated that CD4(+)CD25(+) T cells are able to influence protective immunity underlying premunition to P. berghei NK65 parasites.

Ultrastructural localization of CD36 in human hepatic sinusoidal lining cells, hepatocytes, human hepatoma (HepG2-A16) cells, and C32 amelanotic melanoma cells. Experimental Parasitology 79, 383-390. CD36 is expressed in the endothelial cells of some human organs, but the ultrastructural localization of this molecule in the sinusoidal lining cells of human liver is not well established. We report the ultrastructural localization of CD36 in the liver using a novel murine monoclonal antibody against CD36, namely MO30, as a primary antibody. Immunocytochemistry by the postembedding method showed that CD36 was localized in endothelial cells of sinusoids and in hepatocyte microvilli protruding into the space of Disse. Moreover, in cultured human hepatoma (HepG2-A16) cells and C32 amelanotic melanoma cells, MO30 reacted with microvilli. Hence, CD36 expressed on these cells may be involved in recognition and/or entry of these cells by malaria sporozoites.

The number and distribution of γδ T cells in spleens from patients who died of cerebral malaria and from rhesus monkeys severely infected with Plasmodium coatneyi were examined by immunocytochemistry. γδ T cells were significantly increased in these spleens. In a rodent malaria model using Plasmodium chabaudi adami, an avirulent strain of murine malaria parasites, the degree of parasitemia appears to be modulated by the number of γδ T cells in the spleen. As parasitemia increases, these T cells increase in number. At some critical point, γδ T cells in collaboration with macrophages and αβ T cells apparently start to clear parasitized erythrocytes from the blood, leading to an abatement of the parasitemia, which is followed by a reduction in the number of γδ T cells. This γδ T cell phenomenon may be responsible for the self-limiting infection in mice.

We studied the brains of rhesus monkeys infected with the primate malaria parasite Plasmodium fragile. Electron microscopy showed that, in these animals, erythrocytes infected with P. fragile undergo sequestration and that parasitized red blood cells adhere to endothelial cells in the cerebral microvessels by means of knobs. Cerebral microvessels with sequestered parasitized red blood cells were shown by immunohistochemical analysis to possess the platelet glycoprotein CD36, thrombospondin, and intracellular adhesion molecule-1. The formation of rosettes also was observed in the cerebral microvessels. In a fashion similar to human cerebral malaria, P. fragile produced neurological symptoms in the animals. Thus, rhesus monkeys infected with P. fragile, like those monkeys infected with Plasmodium coatneyi, can be used as a primate model to study human cerebral malaria.

Summary Previous experiments have shown that the infectivity of virulent trypomastigotes in L-cell culture was much more intensive not only for macrophages but also for newly cultured fibroblasts from the mouse heart as compared to low-virulent trypomastigotes in L-cell culture which had been derived from the same strain. On the other hand, the infectivity of virulent trypomastigotes for L-cells was less intensive. In the present work, new fibroblasts derived from muscles, skin, heart and kidney of newborn mice and established fibroblasts from mouse muscles were examined in terms of their susceptibility to trypomastigotes of different virulence. A slight difference of susceptibility to virulent trypomastigotes among new fibroblasts from different sources was found, i. e. fibroblasts derived from muscles and skin were more susceptible than those from heart and kidney. Growth of low-virulent trypomastigotes was suppressed in all new fibroblasts. This suppression was explained by the result that amastigote proliferation of a low-virulent strain was much slower in new fibroblasts from mouse muscles in spite of a similar penetrative ability of both kinds of trypomastigotes. Furthermore, a slight decrease in the susceptibility of new fibroblasts to virulent trypomastigotes was found depending on the length of time fibroblasts had been maintained.