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Although 'Candidatus Mycoplasma haematomacacae' (formerly known as 'Candidatus Mycoplasma haemomacaque') has been reported on extensively in macaques from Thailand, the USA, Japan, and Brazil, its genetic characterization has primarily been restricted to the 16S rRNA sequences with no exploration on multi-locus sequence analysis. The primary goal of this study was to characterize 'Ca. M. haematomacacae' among Thai macaques based on multiple genetic markers. Between April 2018 and November 2021, blood samples were taken from 580 free-ranging macaques (560 Macaca fascicularis and 20 Macaca nemestrina) in 15 locations encompassing 10 provinces throughout Thailand. Using the conventional PCR assay targeting the 16S ribosomal RNA (16S rRNA) gene, 338 out of 580 macaques (58.27 %) tested hemoplasma-positive. Of these, 40 positive samples were further subjected to DNA sequencing, and all were identified as 'Ca. M. haematomacacae'. Subsequently, the partial nucleotide sequences of 23S ribosomal RNA (23S rRNA) and RNase P RNA (rnpB) genes of this particular hemoplasma species were amplified through nested PCR assay. The analysis of multi-locus genetic markers revealed that the 23S rRNA and rnpB sequences exhibited higher levels of genetic diversity than the 16S rRNA sequences. Furthermore, the 16S rRNA analyses demonstrated that 'Ca. M. haematomacacae' infecting Old World monkeys (Macaca spp.) was most closely related to hemotropic Mycoplasma spp. in black-capped capuchins (Sapajus apella) and Marcgrave's capuchins (Sapajus flavius) from Brazil, as well as establishing a common ancestor clade with hemotropic Mycoplasma spp. from the Neotropical bats in Belize and Peru and an Old World bat in Spain. The 23S rRNA analyses likewise evidenced that 'Ca. M. haematomacacae' formed a sister clade with hemotropic Mycoplasma spp. in Neotropical bats from Belize and Panama. Thus, the present findings, based on multi-locus sequence analysis, suggest a potential origin of 'Ca. M. haematomacacae' from Neotropical and Old World bats. To the best of the authors' knowledge, this study provided the largest dataset so far of multi-locus genetic sequences of 'Ca. M. haematomacacae' isolated from Thai macaques and enhanced the accuracy of phylogenetic analyses, providing insights into their origins among hemotropic Mycoplasma spp. discovered worldwide., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. We also confirm that our work is original and has not been submitted elsewhere., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Despite the natural occurrences of human infections by Plasmodium knowlesi, P. cynomolgi, P. inui, and P. fieldi in Thailand, investigating the prevalence and genetic diversity of the zoonotic simian malaria parasites in macaque populations has been limited to certain areas. To address this gap, a total of 560 long-tailed macaques (Macaca fascicularis) and 20 southern pig-tailed macaques (M. nemestrina) were captured from 15 locations across 10 provinces throughout Thailand between 2018 and 2021 for investigation of malaria, as were 15 human samples residing in two simian-malaria endemic provinces, namely Songkhla and Satun, who exhibited malaria-like symptoms. Using PCR techniques targeting the mitochondrial cytb and cox1 genes coupled with DNA sequencing, 40 long-tailed macaques inhabiting five locations had mono-infections with one of the three simian malaria species. Most of the positive cases of macaque were infected with P. inui (32/40), while infections with P. cynomolgi (6/40) and P. knowlesi (2/40) were less common and confined to specific macaque populations. Interestingly, all 15 human cases were mono-infected with P. knowlesi, with one of them residing in an area with two P. knowlesi-infected macaques. Nucleotide sequence analysis showed a high level of genetic diversity in P. inui, while P. cynomolgi and P. knowlesi displayed limited genetic diversity. Phylogenetic and haplotype network analyses revealed that P. inui in this study was closely related to simian and Anopheles isolates from Peninsular Malaysia, while P. cynomolgi clustered with simian and human isolates from Asian countries. P. knowlesi, which was found in both macaques and humans in this study, was closely related to isolates from macaques, humans, and An. hackeri in Peninsular Malaysia, suggesting a sylvatic transmission cycle extending across these endemic regions. This study highlights the current hotspots for zoonotic simian malaria and sheds light on the genetic characteristics of recent isolates in both macaques and human residents in Thailand., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest. We also confirm that our work is original and has not been submitted elsewhere. We, (Copyright © 2023 Elsevier B.V. All rights reserved.)

Plasmodium parasites cause malaria with disease outcomes ranging from mild illness to deadly complications such as severe malarial anemia (SMA), pulmonary edema, acute renal failure, and cerebral malaria. In young children, SMA often requires blood transfusion and is a major cause of hospitalization. Malaria parasite infection leads to the destruction of infected and noninfected erythrocytes as well as dyserythropoiesis; however, the mechanism of dyserythropoiesis accompanied by splenomegaly is not completely understood. Using Plasmodium yoelii yoelii 17XNL as a model, we show that both a defect in erythroblastic island (EBI) macrophages in supporting red blood cell (RBC) maturation and the destruction of reticulocytes/RBCs by the parasites contribute to SMA and splenomegaly. After malaria parasite infection, the destruction of both infected and noninfected RBCs stimulates extramedullary erythropoiesis in mice. The continuous decline of RBCs stimulates active erythropoiesis and drives the expansion of EBIs in the spleen, contributing to splenomegaly. Phagocytosis of malaria parasites by macrophages in the bone marrow and spleen may alter their functional properties and abilities to support erythropoiesis, including reduced expression of the adherence molecule CD169 and inability to support erythroblast differentiation, particularly RBC maturation in vitro and in vivo. Therefore, macrophage dysfunction is a key mechanism contributing to SMA. Mitigating and/or alleviating the inhibition of RBC maturation may provide a treatment strategy for SMA.

COVID-19 is a respiratory disease that can spread rapidly. Controlling the spread through vaccination is one of the measures for activating immunization that helps to reduce the number of infected people. Different types of vaccines are effective in preventing and alleviating the symptoms of the disease in different ways. In this study, a mathematical model, SVIHR , was developed to assess the behavior of disease transmission in Thailand by considering the vaccine efficacy of different vaccine types and the vaccination rate. The equilibrium points were investigated and the basic reproduction number R0 was calculated using a next-generation matrix to determine the stability of the equilibrium. We found that the disease-free equilibrium point was asymptotically stable if, and only if, R0<1, and the endemic equilibrium was asymptotically stable if, and only if, R0>1. The simulation results and the estimation of the parameters applied to the actual data in Thailand are reported. The sensitivity of parameters related to the basic reproduction number was compared with estimates of the effectiveness of pandemic controls. The simulations of different vaccine efficacies for different vaccine types were compared and the average mixing of vaccine types was reported to assess the vaccination policies. Finally, the trade-off between the vaccine efficacy and the vaccination rate was investigated, resulting in the essentiality of vaccine efficacy to restrict the spread of COVID-19.

Unlike malaria parasites in humans, non-human primates, rodents, and birds, ungulate malaria parasites and their vectors have received little attention. As a result, understanding of the hosts, vectors, and biology of ungulate malaria parasites has remained limited. In this study, we aimed to identify the vectors of the goat malaria parasite Plasmodium caprae. A total of 1019 anopheline and 133 non-anopheline mosquitoes were collected from goat farms in Thailand, where P. caprae-infected goats were discovered. Anopheline mosquitoes were identified using molecular biological methods that target the cytochrome c oxidase subunit 1 (cox1), the cytochrome c oxidase subunit 2 (cox2) genes, and the internal transcribed spacer 2 (ITS2) region. Pool and individual mosquitoes were tested for P. caprae using the head-thorax parts that contain the salivary glands, with primers targeting three genetic markers including cytochrome b, cytochrome c oxidase subunit 1, and 18S small subunit ribosomal RNA genes. Additionally, goat blood samples were collected concurrently with mosquito surveys and screened to determine the status of malaria infection. This study revealed nine mosquito species belonging to six groups on goat farms, including Hyrcanus, Barbirostris, Subpictus, Funestus, Tessellatus, and Annularis. The DNA of P. caprae was detected in Anopheles subpictus and Anopheles aconitus. This is the first time An. subpictus and An. aconitus have been implicated as probable vectors of P. caprae., (© 2023. The Author(s).)

Genetic mapping has been widely employed to search for genes linked to phenotypes/traits of interest. Because of the ease of maintaining rodent malaria parasites in laboratory mice, many genetic crosses of rodent malaria parasites have been performed to map the parasite genes contributing to malaria parasite development, drug resistance, host immune response, and disease pathogenesis. Drs. Richard Carter, David Walliker, and colleagues at the University of Edinburgh, UK, were the pioneers in developing the systems for genetic mapping of malaria parasite traits, including characterization of genetic markers to follow the inheritance and recombination of parasite chromosomes and performing the first genetic cross using rodent malaria parasites. Additionally, many genetic crosses of inbred mice have been performed to link mouse chromosomal loci to the susceptibility to malaria parasite infections. In this chapter, we review and discuss past and recent advances in genetic marker development, performing genetic crosses, and genetic mapping of both parasite and host genes. Genetic mappings using models of rodent malaria parasites and inbred mice have contributed greatly to our understanding of malaria, including parasite development within their hosts, mechanism of drug resistance, and host-parasite interaction., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Published by Elsevier B.V.)

Rodent malaria parasites have been widely used in all aspects of malaria research to study parasite development within rodent and insect hosts, drug resistance, disease pathogenesis, host immune response, and vaccine efficacy. Rodent malaria parasites were isolated from African thicket rats and initially characterized by scientists at the University of Edinburgh, UK, particularly by Drs. Richard Carter, David Walliker, and colleagues. Through their efforts and elegant work, many rodent malaria parasite species, subspecies, and strains are now available. Because of the ease of maintaining these parasites in laboratory mice, genetic crosses can be performed to map the parasite and host genes contributing to parasite growth and disease severity. Recombinant DNA technologies are now available to manipulate the parasite genomes and to study gene functions efficiently. In this chapter, we provide a brief history of the isolation and species identification of rodent malaria parasites. We also discuss some recent studies to further characterize the different developing stages of the parasites including parasite genomes and chromosomes. Although there are differences between rodent and human malaria parasite infections, the knowledge gained from studies of rodent malaria parasites has contributed greatly to our understanding of and the fight against human malaria., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2022 Elsevier B.V. All rights reserved.)

At present, a large number of people worldwide have been infected by coronavirus 2019 (COVID-19). When the outbreak of the COVID-19 pandemic begins in a country, its impact is disastrous to both the country and its neighbors. In early 2020, the spread of COVID-19 was associated with global aviation. More recently, COVID-19 infections due to illegal or undocumented immigration have played a significant role in spreading the disease in Southeast Asia countries. Therefore, the spread of COVID-19 of all countries' border should be curbed. Many countries closed their borders to all nations, causing an unprecedented decline in global travel, especially cross-border travel. This restriction affects social and economic trade-offs. Therefore, immigration policies are essential to control the COVID-19 pandemic. To understand and simulate the spread of the disease under different immigration conditions, we developed a novel mathematical model called the Legal immigration and Undocumented immigration from natural borders for Susceptible-Infected-Hospitalized and Recovered people (LUSIHR). The purpose of the model was to simulate the number of infected people under various policies, including uncontrolled, fully controlled, and partially controlled countries. The infection rate was parameterized using the collected data from the Department of Disease Control, Ministry of Public Health, Thailand. We demonstrated that the model possesses nonnegative solutions for favorable initial conditions. The analysis of numerical experiments showed that we could control the virus spread and maintain the number of infected people by increasing the control rate of undocumented immigration across the unprotected natural borders. Next, the obtained parameters were used to visualize the effect of the control rate on immigration at the natural border. Overall, the model was well-suited to explaining and building the simulation. The parameters were used to simulate the trends in the number of people infected from COVID-19., Competing Interests: The authors have declared that no competing interests exist.

Background: Vaccines against the sexual stages of the malarial parasite Plasmodium falciparum are indispensable for controlling malaria and abrogating the spread of drug-resistant parasites. Pfs25, a surface antigen of the sexual stage of P. falciparum, is a leading candidate for transmission-blocking vaccine development. While clinical trials have reported that Pfs25-based vaccines are safe and effective in inducing transmission-blocking antibodies, the extent of the genetic diversity of Pfs25 in malaria endemic populations has rarely been studied. Thus, this study aimed to investigate the global diversity of Pfs25 in P. falciparum populations., Methods: A database of 307 Pfs25 sequences of P. falciparum was established. Population genetic analyses were performed to evaluate haplotype and nucleotide diversity, analyze haplotypic distribution patterns of Pfs25 in different geographical populations, and construct a haplotype network. Neutrality tests were conducted to determine evidence of natural selection. Homology models of the Pfs25 haplotypes were constructed, subjected to molecular dynamics (MD), and analyzed in terms of flexibility and percentages of secondary structures., Results: The Pfs25 gene of P. falciparum was found to have 11 unique haplotypes. Of these, haplotype 1 (H1) and H2, the major haplotypes, represented 70% and 22% of the population, respectively, and were dominant in Asia, whereas only H1 was dominant in Africa, Central America, and South America. Other haplotypes were rare and region-specific, resulting in unique distribution patterns in different geographical populations. The diversity in Pfs25 originated from ten single-nucleotide polymorphism (SNP) loci located in the epidermal growth factor (EGF)-like domains and anchor domain. Of these, an SNP at position 392 (GGA/GCA), resulting in amino acid substitution 131 (Gly/Ala), defined the two major haplotypes. The MD results showed that the structures of H1 and H2 variants were relatively similar. Limited polymorphism in Pfs25 could likely be due to negative selection., Conclusions: The study successfully established a Pfs25 sequence database that can become an essential tool for monitoring vaccine efficacy, designing assays for detecting malaria carriers, and conducting epidemiological studies of P. falciparum. The discovery of the two major haplotypes, H1 and H2, and their conserved structures suggests that the current Pfs25-based vaccines could be used globally for malaria control., (© 2021. The Author(s).)

Erythrocyte-binding-like (EBL) proteins are known to play an important role in malaria parasite invasion of red blood cells (RBCs); however, any roles of EBL proteins in regulating host immune responses remain unknown. Here, we show that Plasmodium yoelii EBL (PyEBL) can shape disease severity by modulating the surface structure of infected RBCs (iRBCs) and host immune responses. We identified an amino acid substitution (a change of C to Y at position 741 [C741Y]) in the protein trafficking domain of PyEBL between isogenic P. yoellii nigeriensis strain N67 and N67C parasites that produce different disease phenotypes in C57BL/6 mice. Exchanges of the C741Y alleles altered parasite growth and host survival accordingly. The C741Y substitution also changed protein processing and trafficking in merozoites and in the cytoplasm of iRBCs, reduced PyEBL binding to band 3, increased phosphatidylserine (PS) surface exposure, and elevated the osmotic fragility of iRBCs, but it did not affect invasion of RBCs in vitro The modified iRBC surface triggered PS-CD36-mediated phagocytosis of iRBCs, host type I interferon (IFN-I) signaling, and T cell differentiation, leading to improved host survival. This study reveals a previously unknown role of PyEBL in regulating host-pathogen interaction and innate immune responses, which may be explored for developing disease control strategies. IMPORTANCE Malaria is a deadly parasitic disease that continues to afflict hundreds of millions of people every year. Infections with malaria parasites can be asymptomatic, with mild symptoms, or fatal, depending on a delicate balance of host immune responses. Malaria parasites enter host red blood cells (RBCs) through interactions between parasite ligands and host receptors, such as erythrocyte-binding-like (EBL) proteins and host Duffy antigen receptor for chemokines (DARC). Plasmodium yoelii EBL (PyEBL) is known to play a role in parasite invasion of RBCs. Here, we show that PyEBL also affects disease severity through modulation of host immune responses, particularly type I interferon (IFN-I) signaling. This discovery assigns a new function to PyEBL and provides a mechanism for developing disease control strategies.

Malaria is a disease with diverse symptoms depending on host immune status and pathogenicity of Plasmodium parasites. The continuous parasite growth within a host suggests mechanisms of immune evasion by the parasite and/or immune inhibition in response to infection. To identify pathways commonly inhibited after malaria infection, we infected C57BL/6 mice with four Plasmodium yoelii strains causing different disease phenotypes and 24 progeny of a genetic cross. mRNAs from mouse spleens day 1 and/or day 4 post infection (p.i.) were hybridized to a mouse microarray to identify activated or inhibited pathways, upstream regulators, and host genes playing an important role in malaria infection. Strong interferon responses were observed after infection with the N67 strain, whereas initial inhibition and later activation of hematopoietic pathways were found after infection with 17XNL parasite, showing unique responses to individual parasite strains. Inhibitions of pathways such as Th1 activation, dendritic cell (DC) maturation, and NFAT immune regulation were observed in mice infected with all the parasite strains day 4 p.i., suggesting universally inhibited immune pathways. As a proof of principle, treatment of N67-infected mice with antibodies against T cell receptors OX40 or CD28 to activate the inhibited pathways enhanced host survival. Controlled activation of these pathways may provide important strategies for better disease management and for developing an effective vaccine.

Development of an effective vaccine is critically needed for the prevention of malaria. One of the key antigens for malaria vaccines is the apical membrane antigen 1 ( AMA -1) of the human malaria parasite Plasmodium falciparum , the surface protein for erythrocyte invasion of the parasite. The gene encoding AMA -1 has been sequenced from populations of P. falciparum worldwide, but the haplotype diversity of the gene in P. falciparum populations in the Greater Mekong Subregion (GMS), including Thailand, remains to be characterized. In the present study, the AMA -1 gene was PCR amplified and sequenced from the genomic DNA of 65 P. falciparum isolates from 5 endemic areas in Thailand. The nearly full-length 1,848 nucleotide sequence of AMA -1 was subjected to molecular analyses, including nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity and neutrality tests. Phylogenetic analysis and pairwise population differentiation ( F st indices) were performed to infer the population structure. The analyses identified 60 single nucleotide polymorphic loci, predominately located in domain I of AMA -1. A total of 31 unique AMA -1 haplotypes were identified, which included 11 novel ones. The phylogenetic tree of the AMA -1 haplotypes revealed multiple clades of AMA -1, each of which contained parasites of multiple geographical origins, consistent with the F in Thailand's borders with Myanmar, Laos and Cambodia. In summary, the study revealed novel haplotypes and population structure needed for the further advancement of st indices indicating genetic homogeneity or gene flow among geographically distinct populations of P. falciparum in Thailand's borders with Myanmar, Laos and Cambodia. In summary, the study revealed novel haplotypes and population structure needed for the further advancement of AMA -1-based malaria vaccines in the GMS.

Background: The glutamate-rich protein (GLURP) of the malaria parasite Plasmodium falciparum is a key surface antigen that serves as a component of a clinical vaccine. Moreover, the GLURP gene is also employed routinely as a genetic marker for malarial genotyping in epidemiological studies. While extensive size polymorphisms in GLURP are well recorded, the extent of the sequence diversity of this gene is rarely investigated. The present study aimed to explore the genetic diversity of GLURP in natural populations of P. falciparum., Results: The polymorphic C-terminal repetitive R2 region of GLURP sequences from 65 P. falciparum isolates in Thailand were generated and combined with the data from 103 worldwide isolates to generate a GLURP database. The collection was comprised of 168 alleles, encoding 105 unique GLURP subtypes, characterized by 18 types of amino acid repeat units (AAU). Of these, 28 GLURP subtypes, formed by 10 AAU types, were detected in P. falciparum in Thailand. Among them, 19 GLURP subtypes and 2 AAU types are described for the first time in the Thai parasite population. The AAU sequences were highly conserved, which is likely due to negative selection. Standard Fst analysis revealed the shared distributions of GLURP types among the P. falciparum populations, providing evidence of gene flow among the different demographic populations., Conclusions: Sequence diversity causing size variations in GLURP in Thai P. falciparum populations were detected, and caused by non-synonymous substitutions in repeat units and some insertion/deletion of aspartic acid or glutamic acid codons between repeat units. The P. falciparum population structure based on GLURP showed promising implications for the development of GLURP-based vaccines and for monitoring vaccine efficacy.

Background: Antigen-detecting rapid diagnostic tests (RDTs) have been recommended by the World Health Organization for use in remote areas to improve malaria case management. Lactate dehydrogenase (LDH) of Plasmodium falciparum is one of the main parasite antigens employed by various commercial RDTs. It has been hypothesized that the poor detection of LDH-based RDTs is attributed in part to the sequence diversity of the gene. To test this, the present study aimed to investigate the genetic diversity of the P. falciparum ldh gene in Thailand and to construct the map of LDH sequence diversity in P. falciparum populations worldwide., Methods: The ldh gene was sequenced for 50 P. falciparum isolates in Thailand and compared with hundreds of sequences from P. falciparum populations worldwide. Several indices of molecular variation were calculated, including the proportion of polymorphic sites, the average nucleotide diversity index (π), and the haplotype diversity index (H). Tests of positive selection and neutrality tests were performed to determine signatures of natural selection on the gene. Mean genetic distance within and between species of Plasmodium ldh was analysed to infer evolutionary relationships., Results: Nucleotide sequences of P. falciparum ldh could be classified into 9 alleles, encoding 5 isoforms of LDH. L1a was the most common allelic type and was distributed in P. falciparum populations worldwide. Plasmodium falciparum ldh sequences were highly conserved, with haplotype and nucleotide diversity values of 0.203 and 0.0004, respectively. The extremely low genetic diversity was maintained by purifying selection, likely due to functional constraints. Phylogenetic analysis inferred the close genetic relationship of P. falciparum to malaria parasites of great apes, rather than to other human malaria parasites., Conclusions: This study revealed the global genetic variation of the ldh gene in P. falciparum, providing knowledge for improving detection of LDH-based RDTs and supporting the candidacy of LDH as a therapeutic drug target.

Infection of mice with strains of Plasmodium yoelii parasites can result in different pathology, but molecular mechanisms to explain this variation are unclear. Here we show that a P. yoelii gene encoding a HECT-like E3 ubiquitin ligase (Pyheul) influences parasitemia and host mortality. We genetically cross two lethal parasites with distinct disease phenotypes, and identify 43 genetically diverse progeny by typing with microsatellites and 9230 single-nucleotide polymorphisms. A genome-wide quantitative trait loci scan links parasite growth and host mortality to two major loci on chromosomes 1 and 7 with LOD (logarithm of the odds) scores = 6.1 and 8.1, respectively. Allelic exchange of partial sequences of Pyheul in the chromosome 7 locus and modification of the gene expression alter parasite growth and host mortality. This study identifies a gene that may have a function in parasite growth, virulence, and host-parasite interaction, and therefore could be a target for drug or vaccine development.Many strains of Plasmodium differ in virulence, but factors that control these distinctions are not known. Here the authors comparatively map virulence loci using the offspring from a P. yoelii YM and N67 genetic cross, and identify a putative HECT E3 ubiquitin ligase that may explain the variance.

Clinical manifestations of malaria infection in vertebrate hosts arise from the multiplication of the asexual stage parasites in the blood, while the gametocytes are responsible for the transmission of the disease. Antimalarial drugs that target the blood stage parasites and transmissible gametocytes are rare, but are essentially needed for the effective control of malaria and for limiting the spread of resistance. Artemisinin and its derivatives are the current first-line antimalarials that are effective against the blood stage parasites and gametocytes, but resistance to artemisinin has now emerged and spread in various malaria endemic areas. Therefore, a novel antimalarial drug, or a new drug combination, is critically needed to overcome this problem. The objectives of this study were to evaluate the efficacy of a relatively new antimalarial compound, tafenoquine (TQ), and a combination of TQ and a low dose of artesunate (ATN) on the in vivo blood stage multiplication, gametocyte development and transmission of the avian malaria parasite Plasmodium gallinaceum to the vector Aedes aegypti. The results showed that a 5-d treatment with TQ alone was unable to clear the blood stage parasites, but was capable of reducing the mortality rate, while TQ monotherapy at a high dose of 30mg/kg was highly effective against the gametocytes and completely blocked the transmission of P. gallinaceum. In addition, the combination therapy of TQ+ATN completely cleared P. gallinaceum blood stages and sped up the gametocyte clearance from chickens, suggesting the synergistic effect of the two drugs. In conclusion, TQ is demonstrated to be effective for limiting avian malaria transmission and may be used in combination with a low dose of ATN for safe and effective treatment., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: An effective malaria vaccine is an urgently needed tool to fight against human malaria, the most deadly parasitic disease of humans. One promising candidate is the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum. This antigenic protein, encoded by the merozoite surface protein (msp-3) gene, is polymorphic and classified according to size into the two allelic types of K1 and 3D7. A recent study revealed that both the K1 and 3D7 alleles co-circulated within P. falciparum populations in Thailand, but the extent of the sequence diversity and variation within each allelic type remains largely unknown., Methods: The msp-3 gene was sequenced from 59 P. falciparum samples collected from five endemic areas (Mae Hong Son, Kanchanaburi, Ranong, Trat and Ubon Ratchathani) in Thailand and analysed for nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity. The gene was also subject to population genetic analysis (F st ) and neutrality tests (Tajima's D, Fu and Li D* and Fu and Li' F* tests) to determine any signature of selection., Results: The sequence analyses revealed eight unique DNA haplotypes and seven amino acid sequence variants, with a haplotype and nucleotide diversity of 0.828 and 0.049, respectively. Neutrality tests indicated that the polymorphism detected in the alanine heptad repeat region of MSP-3 was maintained by positive diversifying selection, suggesting its role as a potential target of protective immune responses and supporting its role as a vaccine candidate. Comparison of MSP-3 variants among parasite populations in Thailand, India and Nigeria also inferred a close genetic relationship between P. falciparum populations in Asia., Conclusion: This study revealed the extent of the msp-3 gene diversity in P. falciparum in Thailand, providing the fundamental basis for the better design of future blood stage malaria vaccines against P. falciparum.

IFN-γ is a major regulator of immune functions and has been shown to induce liver-stage Plasmodium elimination both in vitro and in vivo. The molecular mechanism responsible for the restriction of liver-stage Plasmodium downstream of IFN-γ remains uncertain, however. Autophagy, a newly described immune defense mechanism, was recently identified as a downstream pathway activated in response to IFN-γ in the control of intracellular infections. We thus hypothesized that the killing of liver-stage malarial parasites by IFN-γ involves autophagy induction. Our results show that whereas IFN-γ treatment of human hepatocytes activates autophagy, the IFN-γ-mediated restriction of liver-stage Plasmodium vivax depends only on the downstream autophagy-related proteins Beclin 1, PI3K, and ATG5, but not on the upstream autophagy-initiating protein ULK1. In addition, IFN-γ enhanced the recruitment of LC3 onto the parasitophorous vacuole membrane (PVM) and increased the colocalization of lysosomal vesicles with P. vivax compartments. Taken together, these data indicate that IFN-γ mediates the control of liver-stage P. vivax by inducing a noncanonical autophagy pathway resembling that of LC3-associated phagocytosis, in which direct decoration of the PVM with LC3 promotes the fusion of P. vivax compartments with lysosomes and subsequent killing of the pathogen. Understanding the hepatocyte response to IFN-γ during Plasmodium infection and the roles of autophagy-related proteins may provide an urgently needed alternative strategy for the elimination of this human malaria.

Detoxification of hemoglobin byproducts or free heme is an essential step and considered potential targets for anti-malaria drug development. However, most of anti-malaria drugs are no longer effective due to the emergence and spread of the drug resistant malaria parasites. Therefore, it is an urgent need to identify potential new targets and even for target combinations for effective malaria drug design. In this work, we reconstructed the metabolic networks of Plasmodium falciparum and human red blood cells for the simulation of steady mass and flux flows of the parasite's metabolites under the blood environment by flux balance analysis (FBA). The integrated model, namely iPF-RBC-713, was then adjusted into two stage-specific metabolic models, which first was for the pathological stage metabolic model of the parasite when invaded the red blood cell without any treatment and second was for the treatment stage of the parasite when a drug acted by inhibiting the hemozoin formation and caused high production rate of heme toxicity. The process of identifying target combinations consisted of two main steps. Firstly, the optimal fluxes of reactions in both the pathological and treatment stages were computed and compared to determine the change of fluxes. Corresponding enzymes of the reactions with zero fluxes in the treatment stage but non-zero fluxes in the pathological stage were predicted as a preliminary list of potential targets in inhibiting heme detoxification. Secondly, the combinations of all possible targets listed in the first step were examined to search for the best promising target combinations resulting in more effective inhibition of the detoxification to kill the malaria parasites. Finally, twenty-three enzymes were identified as a preliminary list of candidate targets which mostly were in pyruvate metabolism and citrate cycle. The optimal set of multiple targets for blocking the detoxification was a set of heme ligase, adenosine transporter, myo-inositol 1-phosphate synthase, ferrodoxim reductase-like protein and guanine transporter. In conclusion, the method has shown an effective and efficient way to identify target combinations which are obviously useful in the development of novel antimalarial drug combinations., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Invading pathogens trigger specific host responses, an understanding of which might identify genes that function in pathogen recognition and elimination. In this study, we performed trans-species expression quantitative trait locus (ts-eQTL) analysis using genotypes of the Plasmodium yoelii malaria parasite and phenotypes of mouse gene expression. We significantly linked 1,054 host genes to parasite genetic loci (LOD score ≥ 3.0). Using LOD score patterns, which produced results that differed from direct expression-level clustering, we grouped host genes that function in related pathways, allowing functional prediction of unknown genes. As a proof of principle, 14 of 15 randomly selected genes predicted to function in type I interferon (IFN-I) responses were experimentally validated using overexpression, small hairpin RNA knockdown, viral infection, and/or infection of knockout mice. This study demonstrates an effective strategy for studying gene function, establishes a functional gene database, and identifies regulators in IFN-I pathways., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian malaria is one of the most common veterinary problems in Southeast Asia. The standard molecular method for detection of the avian malaria parasite involves the phenol-chloroform extraction of parasite genomic (g)DNA followed by the amplification of parasite gDNA using polymerase chain reaction (PCR). However, the phenol-chloroform extraction method is time-consuming and requires large amounts of samples and toxic organic solvents, thereby limiting its applications for parasite detection in the field. This study aimed to compare the performance of chelex-100 resin and phenol/chloroform extraction methods for the extraction of Plasmodium gallinaceum gDNA from whole avian blood that had been dried on filter papers (a common field sampling method). The specificity and sensitivity of PCR assays for P. gallinaceum cytochrome B (cytb) and cytochrome oxidase subunit I (coxI) gene fragments (544 and 588bp, respectively) were determined, and found to be more sensitive with gDNA extracted by the chelex-100 resin method than with the phenol/chloroform method. These PCR assays were also performed to detect P. gallinaceum in 29 blood samples dried on filter papers from domestic chickens in a malaria endemic area, where the reliable identification of seven field isolates of P. gallinaceum was obtained with an accuracy of 100%. The analysis of cytb and coxI gene nucleotide sequences revealed the existence of at least two genetically distinct populations of P. gallinaceum in Thailand, both of which differed from the reference strain 8A of P. gallinaceum. In conclusion, the chelex-100 resin extraction method is a simple and sensitive method for isolating gDNA from whole avian blood dried on filter paper. Genomic DNA extracted by the chelex method could subsequently be applied for the PCR-based detection of P. gallinaceum and DNA sequencing. Our PCR assays provide a reliable diagnostic tool for molecular epidemiological studies of P. gallinaceum infections in domestic chickens and wild birds., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Merozoite surface proteins (MSPs) of malaria parasites play critical roles during the erythrocyte invasion and so are potential candidates for malaria vaccine development. However, because MSPs are often under strong immune selection, they can exhibit extensive genetic diversity. The gene encoding the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum displays 2 allelic types, K1 and 3D7. In Thailand, the allelic frequency of the P. falciparum msp-3 gene was evaluated in a single P. falciparum population in Tak at the Thailand and Myanmar border. However, no study has yet looked at the extent of genetic diversity of the msp-3 gene in P. falciparum populations in other localities. Here, we genotyped the msp-3 alleles of 63 P. falciparum samples collected from 5 geographical populations along the borders of Thailand with 3 neighboring countries (Myanmar, Laos, and Cambodia). Our study indicated that the K1 and 3D7 alleles coexisted, but at different proportions in different Thai P. falciparum populations. K1 was more prevalent in populations at the Thailand-Myanmar and Thailand-Cambodia borders, whilst 3D7 was more prevalent at the Thailand-Laos border. Global analysis of the msp-3 allele frequencies revealed that proportions of K1 and 3D7 alleles of msp-3 also varied in different continents, suggesting the divergence of malaria parasite populations. In conclusion, the variation in the msp-3 allelic patterns of P. falciparum in Thailand provides fundamental knowledge for inferring the P. falciparum population structure and for the best design of msp-3 based malaria vaccines.

Unlabelled: One unique feature of malaria parasites is the differential transcription of structurally distinct rRNA (rRNA) genes at different developmental stages: the A-type genes are transcribed mainly in asexual stages, whereas the S-type genes are expressed mostly in sexual or mosquito stages. Conclusive functional evidence of different rRNAs in regulating stage-specific parasite development, however, is still absent. Here we performed genetic crosses of Plasmodium yoelii parasites with one parent having an oocyst development defect (ODD) phenotype and another producing normal oocysts to identify the gene(s) contributing to the ODD. The parent with ODD--characterized as having small oocysts and lacking infective sporozoites--was obtained after introduction of a plasmid with a green fluorescent protein gene into the parasite genome and subsequent passages in mice. Quantitative trait locus analysis of genome-wide microsatellite genotypes of 48 progeny from the crosses linked an ~200-kb segment on chromosome 6 containing one of the S-type genes (D-type small subunit rRNA gene [D-ssu]) to the ODD. Fine mapping of the plasmid integration site, gene expression pattern, and gene knockout experiments demonstrated that disruption of the D-ssu gene caused the ODD phenotype. Interestingly, introduction of the D-ssu gene into the same parasite strain (self), but not into a different subspecies, significantly affected or completely ablated oocyst development, suggesting a stage- and subspecies (strain)-specific regulation of oocyst development by D-ssu. This study demonstrates that P. yoelii D-ssu is essential for normal oocyst and sporozoite development and that variation in the D-ssu sequence can have dramatic effects on parasite development., Importance: Malaria parasites are the only known organisms that express structurally distinct rRNA genes at different developmental stages. The differential expression of these genes suggests that they play unique roles during the complex life cycle of the parasites. Conclusive functional proof of different rRNAs in regulating parasite development, however, is still absent or controversial. Here we functionally demonstrate for the first time that a stage-specifically expressed D-type small-subunit rRNA gene (D-ssu) is essential for oocyst development of the malaria parasite Plasmodium yoelii in the mosquito. This study also shows that variations in D-ssu sequence and/or the timing of transcription may have profound effects on parasite oocyst development. The results show that in addition to protein translation, rRNAs of malaria parasites also regulate parasite development and differentiation in a strain-specific manner, which can be explored for controlling parasite transmission., (Copyright © 2015 Qi et al.)

In the absence of vaccines, chemotherapy is an effective and economical way for controlling malaria. Development of anti-malarial drugs that target pathogenic blood stage parasites and gametocytes is preferable for the treatment as it can alleviate the host's morbidity and mortality and block transmission of the Plasmodium parasite. Recently, our laboratory has developed an in vivo transmission blocking assay that involves administration of 7 consecutive daily doses of a test compound into domestic chickens (Gallus gallus domesticus) infected with the avian malaria parasite Plasmodium gallinaceum with 10% parasitaemia and 1% gametocytaemia. To compromise the cost and time for artesunate (ATN) treatment, this study aimed to investigate effects of a 5-day consecutive administration of 10 milligrams per kilogram (mg/kg) ATN on P. gallinaceum infection in chickens and transmission to two natural vectors, Aedes aegypti and Culex quinquefasciatus. Our study showed that the treatment with 10 mg/kg ATN for 7 days, but not 5 days, completely eliminated blood stage infections, prevented recrudescence and blocked gametocyte production and transmission of P. gallinaceum to its vectors, thereby confirming the potent schizontocidal and gametocytocidal activities of ATN. This regimen should be further evaluated in field trials., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The rodent malaria parasite Plasmodium yoelii is an important model for studying malaria immunity and pathogenesis. One approach for studying malaria disease phenotypes is genetic mapping, which requires typing a large number of genetic markers from multiple parasite strains and/or progeny from genetic crosses. Hundreds of microsatellite (MS) markers have been developed to genotype the P. yoelii genome; however, typing a large number of MS markers can be labor intensive, time consuming, and expensive. Thus, development of high-throughput genotyping tools such as DNA microarrays that enable rapid and accurate large-scale genotyping of the malaria parasite will be highly desirable. In this study, we sequenced the genomes of two P. yoelii strains (33X and N67) and obtained a large number of single nucleotide polymorphisms (SNPs). Based on the SNPs obtained, we designed sets of oligonucleotide probes to develop a microarray that could interrogate ∼11,000 SNPs across the 14 chromosomes of the parasite in a single hybridization. Results from hybridizations of DNA samples of five P. yoelii strains or cloned lines (17XNL, YM, 33X, N67 and N67C) and two progeny from a genetic cross (N67×17XNL) to the microarray showed that the array had a high call rate (∼97%) and accuracy (99.9%) in calling SNPs, providing a simple and reliable tool for typing the P. yoelii genome. Our data show that the P. yoelii genome is highly polymorphic, although isogenic pairs of parasites were also detected. Additionally, our results indicate that the 33X parasite is a progeny of 17XNL (or YM) and an unknown parasite. The highly accurate and reliable microarray developed in this study will greatly facilitate our ability to study the genetic basis of important traits and the disease it causes., (Published by Elsevier B.V.)

Background: The 19-kDa C-terminal region of the merozoite surface protein-1 of the human malaria parasite Plasmodium falciparum (PfMSP-119) constitutes the major component on the surface of merozoites and is considered as one of the leading candidates for asexual blood stage vaccines. Because the protein exhibits a level of sequence variation that may compromise the effectiveness of a vaccine, the global sequence diversity of PfMSP-119 has been subjected to extensive research, especially in malaria endemic areas. In Thailand, PfMSP-119 sequences have been derived from a single parasite population in Tak province, located along the Thailand-Myanmar border, since 1995. However, the extent of sequence variation and the spatiotemporal patterns of the MSP-119 haplotypes along the Thai borders with Laos and Cambodia are unknown., Methods: Sixty-three isolates of P. falciparum from five geographically isolated populations along the Thai borders with Myanmar, Laos and Cambodia in three transmission seasons between 2002 and 2008 were collected and culture-adapted. The msp-1 gene block 17 was sequenced and analysed for the allelic diversity, frequency and distribution patterns of PfMSP-119 haplotypes in individual populations. The PfMSP-119 haplotype patterns were then compared between parasite populations to infer the population structure and genetic differentiation of the malaria parasite., Results: Five conserved polymorphic positions, which accounted for five distinct haplotypes, of PfMSP-119 were identified. Differences in the prevalence of PfMSP-119 haplotypes were detected in different geographical regions, with the highest levels of genetic diversity being found in the Kanchanaburi and Ranong provinces along the Thailand-Myanmar border and Trat province located at the Thailand-Cambodia border. Despite this variability, the distribution patterns of individual PfMSP-119 haplotypes seemed to be very similar across the country and over the three malarial transmission seasons, suggesting that gene flow may operate between parasite populations circulating in Thailand and the three neighboring countries., Conclusion: The major MSP-119 haplotypes of P. falciparum populations in all endemic populations during three transmission seasons in Thailand were identified, providing basic information on the common haplotypes of MSP-119 that is of use for malaria vaccine development and inferring the population structure of P. falciparum populations in Thailand.

Malaria infection triggers vigorous host immune responses; however, the parasite ligands, host receptors, and the signaling pathways responsible for these reactions remain unknown or controversial. Malaria parasites primarily reside within RBCs, thereby hiding themselves from direct contact and recognition by host immune cells. Host responses to malaria infection are very different from those elicited by bacterial and viral infections and the host receptors recognizing parasite ligands have been elusive. Here we investigated mouse genome-wide transcriptional responses to infections with two strains of Plasmodium yoelii (N67 and N67C) and discovered differences in innate response pathways corresponding to strain-specific disease phenotypes. Using in vitro RNAi-based gene knockdown and KO mice, we demonstrated that a strong type I IFN (IFN-I) response triggered by RNA polymerase III and melanoma differentiation-associated protein 5, not Toll-like receptors (TLRs), binding of parasite DNA/RNA contributed to a decline of parasitemia in N67-infected mice. We showed that conventional dendritic cells were the major sources of early IFN-I, and that surface expression of phosphatidylserine on infected RBCs might promote their phagocytic uptake, leading to the release of parasite ligands and the IFN-I response in N67 infection. In contrast, an elevated inflammatory response mediated by CD14/TLR and p38 signaling played a role in disease severity and early host death in N67C-infected mice. In addition to identifying cytosolic DNA/RNA sensors and signaling pathways previously unrecognized in malaria infection, our study demonstrates the importance of parasite genetic backgrounds in malaria pathology and provides important information for studying human malaria pathogenesis., Competing Interests: The authors declare no conflict of interest.

Infection and transmission of the avian malaria parasite Plasmodium gallinaceum in domestic chickens is associated with high economic burden and presents a major challenge to poultry industry in South East Asia. Development of drugs targeting both asexual blood stage parasites and sexual stages of the avian malarias will be beneficial for malaria treatment and eradication. However, current drugs recommended for treatment of the avian malaria parasites target specifically the asexual blood stage parasites, but have little or no impact to the gametocytes, the major target for development of transmission-blocking strategies. In the present work, we established a simple procedure to evaluate gametocytocidal and transmission blocking activities in a P. gallinaceum-avian model. The assays involved administration of seven consecutive daily doses of test compounds into P. gallinaceum-infected chickens with 10% parasitaemia and 1% gametocytaemia. Our studies indicated that intramuscular injection with seven daily low doses (the minimum effective dose of 10mg/kg) of artesunate blocked the gametocyte production and transmission to the mosquito vector Aedes aegypti. This assay can be further applicable for testing new compounds against P. gallinaceum and for other parasitic protozoa infecting birds., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Malaria is a deadly infectious disease in many tropical and subtropical countries. Previous efforts to eradicate malaria have failed, largely due to the emergence of drug-resistant parasites, insecticide-resistant mosquitoes and, in particular, the lack of drugs or vaccines to block parasite transmission. ATP-binding cassette (ABC) transporters are known to play a role in drug transport, metabolism, and resistance in many organisms, including malaria parasites. To investigate whether a Plasmodium falciparum ABC transporter (Pf14&#95;0244 or PfABCG2) modulates parasite susceptibility to chemical compounds or plays a role in drug resistance, we disrupted the gene encoding PfABCG2, screened the recombinant and the wild-type 3D7 parasites against a library containing 2,816 drugs approved for human or animal use, and identified an antihistamine (ketotifen) that became less active against the PfABCG2-disrupted parasite in culture. In addition to some activity against asexual stages and gametocytes, ketotifen was highly potent in blocking oocyst development of P. falciparum and the rodent parasite Plasmodium yoelii in mosquitoes. Tests of structurally related tricyclic compounds identified additional compounds with similar activities in inhibiting transmission. Additionally, ketotifen appeared to have some activity against relapse of Plasmodium cynomolgi infection in rhesus monkeys. Further clinical evaluation of ketotifen and related compounds, including synthetic new derivatives, in blocking malaria transmission may provide new weapons for the current effort of malaria eradication.

Genetic cross is a powerful tool for studying malaria genes contributing to drug resistance, parasite development, and pathogenesis. Cloning and identification of recombinant progeny (RP) is laborious and expensive, especially when a large proportion of progeny derived from self-fertilization are present in the uncloned progeny of a genetic cross. Since the frequency of cross-fertilization affects the number of recombinant progeny in a genetic cross, it is important to optimize the procedure of a genetic cross to maximize the cross-fertilization. Here we investigated the factors that might influence the chances of obtaining RP from a genetic cross and showed that different Plasmodium yoelii strains/subspecies/clones had unique abilities in producing oocysts in a mosquito midgut. When a genetic cross is performed between two parents producing different numbers of functional gametocytes, the ratio of parental parasites must be adjusted to improve the chance of obtaining RP. An optimized parental ratio could be established based on oocyst counts from single infection of each parent before crossing experiments, which may reflect the efficiency of gametocyte production and/or fertilization. The timing of progeny cloning is also important; cloning of genetic cross progeny from mice directly infected with sporozoites (vs. frozen blood after needle passage) at a time when parasitemia is low (usually <1%) could improve the chance of obtaining RP. This study provides an optimized protocol for efficiently cloning RPs from a genetic cross of malaria parasites., (Published by Elsevier Inc.)

Malaria remains a devastating disease largely because of widespread drug resistance. New drugs and a better understanding of the mechanisms of drug action and resistance are essential for fulfilling the promise of eradicating malaria. Using high-throughput chemical screening and genome-wide association analysis, we identified 32 highly active compounds and genetic loci associated with differential chemical phenotypes (DCPs), defined as greater than or equal to fivefold differences in half-maximum inhibitor concentration (IC(50)) between parasite lines. Chromosomal loci associated with 49 DCPs were confirmed by linkage analysis and tests of genetically modified parasites, including three genes that were linked to 96% of the DCPs. Drugs whose responses mapped to wild-type or mutant pfcrt alleles were tested in combination in vitro and in vivo, which yielded promising new leads for antimalarial treatments.

Plasmodium yoelii is an excellent model for studying malaria pathogenesis that is often intractable to investigate using human parasites; however, genetic studies of the parasite have been hindered by lack of genome-wide linkage resources. Here, we performed 14 genetic crosses between three pairs of P. yoelii clones/subspecies, isolated 75 independent recombinant progeny from the crosses, and constructed a high-resolution linkage map for this parasite. Microsatellite genotypes from the progeny formed 14 linkage groups belonging to the 14 parasite chromosomes, allowing assignment of sequence contigs to chromosomes. Growth-related virulent phenotypes from 25 progeny of one of the crosses were significantly associated with a major locus on chromosome 13 and with two secondary loci on chromosomes 7 and 10. The chromosome 10 and 13 loci are both linked to day 5 parasitemia, and their effects on parasite growth rate are independent but additive. The locus on chromosome 7 is associated with day 10 parasitemia. The chromosome 13 locus spans ~220 kb of DNA containing 51 predicted genes, including the P. yoelii erythrocyte binding ligand, in which a C741Y substitution in the R6 domain is implicated in the change of growth rate. Similarly, the chromosome 10 locus spans ~234 kb with 71 candidate genes, containing a member of the 235-kDa rhoptry proteins (Py235) that can bind to the erythrocyte surface membrane. Atypical virulent phenotypes among the progeny were also observed. This study provides critical tools and information for genetic investigations of virulence and biology of P. yoelii.

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents and humans. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh. Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Despite many decades of research, no registered vaccine against the pathogenic blood stages of the malaria parasite exists, translating into the loss of many hundreds of thousands of young lives each year in tropical Africa. Although many parasite proteins have been shown to induce immune responses in the host, proof for their induction of protective immunity is still lacking. We previously reported a novel genetic approach called linkage group selection (LGS) for rapid identification of target antigens of strain-specific protective immunity (SSPI) against malaria. In preliminary LGS experiments, we crossed two genetically distinct strains of Plasmodium chabaudi chabaudi and subjected their progeny to selection in strain-specifically immunised mice, measuring the effects of SSPI selection with low coverage/resolution genetic markers. In the present study, through application of high coverage/resolution, single nucleotide polymorphism (SNP) markers spanning all 14 parasite chromosomes, we analysed 35 SSPI selection events on different populations of progeny parasites. Here we report a comprehensive high resolution genome-wide analysis of the effects of strain-specific immune selection on blood stage parasites. Our analyses consistently identify a single genomic region spanning approximately 79kb on chromosome 8 as the region controlling SSPI. Within this region, one gene (that of merozoite surface protein 1, MSP-1) accounted for >60% of genetic polymorphism and was most frequently under greatest reduction under SSPI. These results, combined with those of an independent LGS analysis of a different genetic cross with different parental strains, demonstrate that more than any other locus, the gene for MSP-1 determines the effect of strain-specific protective immunity against malaria in these host-parasite combinations. Our results provide unique insight into the precise timing of the parasite killing immune response against progeny parasites carrying specific alleles of MSP-1; these findings pave the way for investigating which part(s) of this highly polymorphic molecule mediate the protective immune response., (2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Variation in the multiplication rate of blood stage malaria parasites is often positively correlated with the severity of the disease they cause. The rodent malaria parasite Plasmodium yoelii yoelii has strains with marked differences in multiplication rate and pathogenicity in the blood. We have used genetic analysis by linkage group selection (LGS) to identify genes that determine differences in multiplication rate. Genetic crosses were generated between genetically unrelated, fast- (17XYM) and slowly multiplying (33XC) clones of P. y. yoelii. The uncloned progenies of these crosses were placed under multiplication rate selection in blood infections in mice. The selected progenies were screened for reduction in intensity of quantitative genetic markers of the slowly multiplying parent. A small number of strongly selected markers formed a linkage group on P. y. yoelii chromosome 13. Of these, that most strongly selected marked the gene encoding the P. yoelii erythrocyte binding ligand (pyebl), which has been independently identified by Otsuki and colleagues [Otsuki H, et al. (2009) Proc Natl Acad Sci USA 106:10.1073/pnas.0811313106] as a major determinant of virulence in these parasites. In an analysis of a previous genetic cross in P. y. yoelii, pyebl alleles of fast- and slowly multiplying parents segregated with the fast and slow multiplication rate phenotype in the cloned recombinant progeny, implying the involvement of the pyebl locus in determining the multiplication rate. Our genome-wide LGS analysis also indicated effects of at least 1 other locus on multiplication rate, as did the findings of Otsuki and colleagues on virulence in P. y. yoelii.

Many of the most commonly studied lines of the rodent malaria parasite Plasmodium yoelii yoelii originated from a single parasite isolate designated 17X. Amongst these lines, however, are parasites that exhibit variation in genotype and phenotype (e.g. growth rate). We describe here the results of a comparative genetic analysis between cloned lines of 17X that differ in growth rate, using nucleotide sequences of specific genes and patterns of genome-wide amplified fragment length polymorphism (AFLP). Our findings indicate that the original stock of 17X comprises two unrelated genotypes. Genotype-1 is represented by parasites with a slow growth phenotype (e.g. 17X (NIMR)) and a fast growth phenotype (e.g. 17XYM). Within this genotype, there are also genomic differences manifest as a small number of AFLP bands that differentiate the fast- and slow-growing lines from each other. The other genotype, genotype-2, is represented only by parasites with a slow growth phenotype (e.g. 17XA).

Protective immunity against blood infections of malaria is partly specific to the genotype, or strain, of the parasites. The target antigens of Strain Specific Protective Immunity are expected, therefore, to be antigenically and genetically distinct in different lines of parasite. Here we describe the use of a genetic approach, Linkage Group Selection, to locate the target(s) of Strain Specific Protective Immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi. In a previous such analysis using the progeny of a genetic cross between P. c. chabaudi lines AS-pyr1 and CB, a location on P. c. chabaudi chromosome 8 containing the gene for merozoite surface protein-1, a known candidate antigen for Strain Specific Protective Immunity, was strongly selected. P. c. chabaudi apical membrane antigen-1, another candidate for Strain Specific Protective Immunity, could not have been evaluated in this cross as AS-pyr1 and CB are identical within the cell surface domain of this protein. Here we use Linkage Group Selection analysis of Strain Specific Protective Immunity in a cross between P. c. chabaudi lines CB-pyr10 and AJ, in which merozoite surface protein-1 and apical membrane antigen-1 are both genetically distinct. In this analysis strain specific immune selection acted strongly on the region of P. c. chabaudi chromosome 8 encoding merozoite surface protein-1 and, less strongly, on the P. c. chabaudi chromosome 9 region encoding apical membrane antigen-1. The evidence from these two independent studies indicates that Strain Specific Protective Immunity in P. c. chabaudi in mice is mainly determined by a narrow region of the P. c. chabaudi genome containing the gene for the P. c. chabaudi merozoite surface protein-1 protein. Other regions, including that containing the gene for P. c. chabaudi apical membrane antigen-1, may be more weakly associated with Strain Specific Protective Immunity in these parasites.