UTR introns, antisense RNA and differentially spliced transcripts between Plasmodium yoelii subspecies

Additional file 1. Evaluation of RNA quality from the two NSM parasite samples in agarose gel (a), and a flow chart of data processing and analysis (b)

Evaluation of drug effects on Toxoplasma gondii nuclear and plastid DNA replication using real-time PCR

Toxoplasma gondii Nicolle and Manceaux, 1908 is a unicellular protozoan that can infect a broad spectrum of organisms including humans. In addition to a nuclear genome, it also carries a circular DNA within a plastid-like organelle (apicoplast) and a linear genome within its mitochondria. The plastid organelle has been shown to be the target of various anti-parasitic drugs or antibiotics. To evaluate the effects of agents on the DNA replication of T. gondii, we tested six drugs (ciprofloxacin, acetylspiramycin, clindamycin, azithromycin, artemether, and sulfadiazine) on the parasite cultured in Hela cells. After drug treatment for 48 h, the parasite growth and DNA replication were evaluated and quantitated using TaqMan real-time quantitative PCR with oligonucleotide primers synthesized based on a gene from the apicoplast genome (ycf24, Genbank accession no. U87145) and a gene from the nuclear genome (uprt, Genbank accession no. U10246). Our results showed that ciprofloxacin was the most effective in inhibiting the replication of the plastid DNA after 48 h drug treatment, with a reduction of 22% in the copy number of the plastid DNA. Artemether was the most effective drug in suppressing the proliferation of tachyzoites. This study also demonstrates that real-time quantitative PCR is a simple and useful technique for monitoring parasite growth and DNA replication

Macrophage migration inhibitory factor homolog from Plasmodium yoelii modulates monocyte recruitment and activation in spleen during infection

Macrophage migration inhibitory factor (MIF) has been shown to be involved in the pathogenesis of severe malaria. Malaria parasites express an MIF homolog that may play a role in regulating host immune responses, and a recent study showed that overexpression of MIF reduced parasitemia in a mouse malaria model. Another recent study showed migration of monocytes to the spleen contributed to the control of blood stage infection. However, there are few papers describing the effect of MIF on monocyte recruitment/activation during the infection. We generated recombinant Plasmodium yoelii MIF (rPyMIF) and investigated its function on purified mouse CD11b(+) cells in vitro and monocyte responses in vivo. The result shows that rPyMIF protein bound to mouse CD11b(+) cells and inhibited their random migration in vitro. On the other hand, rPyMIF did not induce cytokine release from the cells directly or modulate lipopolysaccharide-induced cytokine release. Mice immunized with rPyMIF showed transient but significantly lower parasitemia than the control mice at day 3 after lethal Py17XL challenge. The total number of CD11b(+) cells in the spleens was significantly higher in rPyMIF-immunized group. Further investigation revealed that there were significantly higher numbers of recruited and activated monocytes in the spleens of rPyMIF immunization group on day 3. These results indicate that PyMIF potentially modulates monocyte recruitment and activation during infection of P. yoelii erythrocytic stages.Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health; National Basic Research Program of China (973 Program) [2007CB513103]; Science Planning Program of Fujian Province [2010 J1008]; 111 Project of Education of China [B06016

Hundreds of microsatellites for genotyping Plasmodium yoelii parasites

Genetic crosses have been employed to study various traits of rodent malaria parasites and to locate loci that contribute to drug resistance, immune protection, and disease virulence. Compared with human malaria parasites, genetic crossing of rodent malaria parasites is more easily performed; however, genotyping methods using microsatellites (MSs) or large-scale single nucleotide polymorphisms (SNPs) that have been widely used in typing Plasmodium falciparum are not available for rodent malaria species. Here we report a genome-wide search of the Plasmodium yoelii yoelii (P. yoelii) genome for simple sequence repeats (SSRs) and the identification of nearly 600 polymorphic MS markers for typing the genomes of P, yoelii and Plasmodium berghei. The MS markers are randomly distributed across the 14 physical chromosomes assembled from genome sequences of three rodent malaria species, although some variations in the numbers of MS expected according to chromosome size exist. The majority of the MS markers are AT-rich repeats, similar to those found in the P. falciparum genome. The MS markers provide an important resource for genotyping, lay a foundation for developing linkage maps, and will greatly facilitate genetic studies of P. yoelii. Published by Elsevier B.V

A new malaria antigen produces partial protection against Plasmodium yoelii challenge

Of all the parasitic diseases, malaria is the number one killer. Despite tremendous efforts in disease control and research, nearly a million people, primarily children, still die from the disease each year, partly due to drug resistance and the lack of an effective vaccine. Many parasite antigens have been identified and evaluated for vaccine development; however, none has been approved for human use. Antigenic variation, complex life cycle, and inadequate understanding of the mechanisms of parasite-host interaction and of host immune response all contribute to the lack of an effective vaccine for malaria control. In a recent search of genome-wide polymorphism in Plasmodium falciparum, several molecules were found to be recognized by sera from patients infected with the P. falciparum parasite. Here, we have expressed a 350-amino acid N terminus from one of the homologous candidate antigen genes from the rodent malaria parasite Plasmodium yoelii (Py01157, a putative dentin phosphorin) in bacteria and evaluated the immune response and protection generated after immunization with the recombinant protein. We showed that the recombinant protein was recognized by sera from both mice and humans infected with malaria parasites. Partial protection was observed after challenge with non-lethal P. yoelii 17XNL but not with the lethal P. yoelii 17XL parasite. Further tests using a full-length protein or the conserved C terminus may provide additional information on whether this protein has the potential for being a malaria vaccine.973 Program of China [2007CB513103]; Science Planning Program of Fujian Province [2010J1008]; 111 Project of Education of China [B06016]; Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Healt

The Gametocytes of Leucocytozoon sabrazesi Infect Chicken Thrombocytes, Not Other Blood Cells.

Leucocytozoon parasites infect a large number of avian hosts, including domestic chicken, and cause significant economical loss to the poultry industry. Although the transmission stages of the parasites were observed in avian blood cells more than a century ago, the specific host cell type(s) that the gametocytes infect remain uncertain. Because all the avian blood cells, including red blood cells (RBCs), are nucleated, and the developing parasites dramatically change the morphology of the infected host cells, it has been difficult to identify Leucocytozoon infected host cell(s). Here we use cell-type specific antibodies to investigate the identities of the host cells infected by Leucocytozoon sabrazesi gametocytes. Anti-RBC antibodies stained RBCs membrane strongly, but not the parasite-infected cells, ruling out the possibility of RBCs being the infected host cells. Antibodies recognizing various leukocytes including heterophils, monocytes, lymphocytes, and macrophages did not stain the infected cells either. Antisera raised against a peptide of the parasite cytochrome B (CYTB) stained parasite-infected cells and some leukocytes, particularly cells with a single round nucleus as well as clear/pale cytoplasm suggestive of thrombocytes. Finally, a monoclonal antibody known to specifically bind chicken thrombocytes also stained the infected cells, confirming that L. sabrazesi gametocytes develop within chicken thrombocytes. The identification of L. sabrazesi infected host cell solves a long unresolved puzzle and provides important information for studying parasite invasion of host cells and for developing reagents to interrupt parasite transmission

Multi-Strain Infections and ‘Relapse’ of <i>Leucocytozoon sabrazesi</i> Gametocytes in Domestic Chickens in Southern China

<div><p><i>Leucocytozoon</i> parasites infect many species of avian hosts, including domestic chicken, and can inflict heavy economic loss to the poultry industry. Although the prevalence and distribution of two <i>Leucocytozoon</i> species (<i>L. sabrazesi</i> and <i>L. caulleryi</i>) have been reported in China previously, there are many questions related to the parasite infection that remain unanswered, including population diversity and transmission dynamics in domestic chickens. Here we surveyed chicken blood samples from seven sites in four provinces of China to identify <i>Leucocytozoon</i> infection, characterized parasite diversity within individual infected hosts and between sampling sites, and investigated the dynamics of gametocytemia in chickens over time. We found high infection rates in three of the seven sites. Clustering parasite sequences of the mitochondrial cytochrome oxidase III (<i>coxIII</i>) and cytochrome b (<i>cytb</i>) genes showed lack of grouping according to geographic origins and individual hosts carrying large numbers of <i>L. sabrazesi</i> strains. Monitoring gametocytemia in blood samples from infected chickens over time showed ‘relapse’ or persistence of low-level gametocytemia for 4–5 months, which could be explored as an <i>in vivo</i> model for testing drugs against liver stages of Apicomplexan parasites. This study provides important information on population diversity and transmission dynamics of <i>L. sabrazesi</i> and for disease control.</p></div

Correlation plot of ratios of minor alleles of <i>coxIII</i> gene segment from two chicken hosts.

<p>A, Plot of allelic ratios from chicken ZZ24. Minor allele ratios from seven individual sequences were plotted against electropherogram signal ratios of PCR product direct sequencing; B, the same as A, but from 10 individual sequences of chicken HC2. The X-axis is the ratios of minor alleles from individual clones, and the Y-axis is the ratios of peak signals [minor peak/(minor+major peak)] from the sequence of direct PCR product sequencing. Positions without two alleles in the bacterial colony sequences are not included.</p

Multi-Strain Infections and 'Relapse' of Leucocytozoon sabrazesi Gametocytes in Domestic Chickens in Southern China

National Science Foundation of China [81201324, 81271858]; Project 111 of the State Bureau of Foreign Experts; Ministry of Education of China [B06016]; Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of HealthLeucocytozoon parasites infect many species of avian hosts, including domestic chicken, and can inflict heavy economic loss to he poultry industry. Although the prevalence and distribution of two Leucocytozoon species (L. sabrazesi and L. caulleryi) have been reported in China previously, there are many questions related to the parasite infection that remain unanswered, including population diversity and transmission dynamics in domestic chickens. Here we surveyed chicken blood samples from even sites in four provinces of China to identify Leucocytozoon infection, characterized Parasite diversity within individual infected hosts and between sampling sites, and investigated the dynamics of gametocytemia in chickens over time. We found high infection rates in three of the seven sites. Clustering parasite sequences of the mitochondrial cytochrome oxidase III (coxIII) and cytochrome b (cytb) genes showed lack of grouping according to geographic origins and individual hosts carrying large numbers of L. sabrazesi strains Monitoring gametocytemia in blood samples from infected showed 'relapse' or persistence of low-level gametocytemia for 4-5 months, which could be explored as sting drugs against liver stages of Apicomplexan parasites. This study provides important information on population diversity and transmission dynamics of L. sabrazesi and for disease control