RTS,S/AS02A malaria vaccine does not induce parasite CSP T cell epitope selection and reduces multiplicity of infection

Objective: The candidate malaria vaccine RTS,S/AS02A is a recombinant protein containing part of the circumsporozoite protein (CSP) sequence of Plasmodium falciparum, linked to the hepatitis B surface antigen and formulated in the proprietary adjuvant system AS02A. In a recent trial conducted in children younger than age five in southern Mozambique, the vaccinedemonstrated significant and sustained efficacy against both infection and clinical disease. In a follow-up study to the main trial, breakthrough infections identified in the trial were examined to determine whether the distribution of csp sequences was affected by the vaccine and to measure the multiplicity of infecting parasite genotypes. Design: P. falciparum DNA from isolates collected during the trial was used for genotype studies. Setting: The main trial was carried out in the Manhiça district, Maputo province, Mozambique, between April 2003 and May 2004. Participants: Children from the two cohorts of the main trial provided parasite isolates as follows: children from Cohort 1 who were admitted to hospital with clinical malaria; children from Cohort 1 who were parasite-positive in a cross-sectional survey at study month 8.5; children from Cohort 2 identified as parasite-positive during follow-up by active detection of infection. Outcome: Divergence of DNA sequence encoding the CSP T cell-epitope region sequence from that of the vaccine sequence was measured in 521 isolates. The number of distinct P. falciparum genotypes was also determined. Results: We found no evidence that parasite genotypes from children in the RTS,S/AS02A arm were more divergent than those receiving control vaccines. For Cohort 1 (survey at studymonth 8.5) and Cohort 2, infections in the vaccine group contained significantly fewer genotypes than those in the control group, (p 1/4 0.035, p 1/4 0.006), respectively, for the two cohorts. This was not the case for children in Cohort 1 who were admitted to hospital (p 1/4 0.478). Conclusions: RTS,S/AS02A did not select for genotypes encoding divergent T cell epitopes in the C-terminal region of CSP in this trial. In both cohorts, there was a modest reduction in the mean number of parasite genotypes harboured by vaccinated children compared with controls, but only among those with asymptomatic infections.

Evolutionary analysis of genes of two pathways involved in placental malaria infection

Placental malaria is a special form of malaria that causes up to 200,000 maternal and infant deaths every year. Previous studies show that two receptor molecules, hyaluronic acid and chondroitin sulphate A, are mediating the adhesion of parasite-infected erythrocytes in the placenta of patients, which is believed to be a key step in the pathogenesis of the disease. In this study, we aimed at identifying sites of malaria-induced adaptation by scanning for signatures of natural selection in 24 genes in the complete biosynthesis pathway of these two receptor molecules. We analyzed a total of 24 Mb of publicly available polymorphism data from the International HapMap project for three human populations with European, Asian and African ancestry, with the African population from a region of presently and historically high malaria prevalence. Using the methods based on allele frequency distributions, genetic differentiation between populations, and on long-range haplotype structure, we found only limited evidence for malaria-induced genetic adaptation in this set of genes in the African population; however, we identified one candidate gene with clear evidence of selection in the Asian population. Although historical exposure to malaria in this population cannot be ruled out, we speculate that it might be caused by other pathogens, as there is growing evidence that these molecules are important receptors in a variety of host-pathogen interactions. We propose to use the present methods in a systematic way to help identify candidate regions under positive selection as a consequence of malaria.

A variant in the gene FUT9 is associated with susceptibility to placental malaria infection

Malaria in pregnancy forms a substantial part of the worldwide burden of malaria, with an estimated annual death toll of up to 200,000 infants, as well as increased maternal morbidity and mortality. Studies of genetic susceptibility to malaria have so far focused on infant malaria, with only a few studies investigating the genetic basis of placental malaria, focusing only on a limited number of candidate genes. The aim of this study therefore was to identify novel host genetic factors involved in placental malaria infection. To this end we carried out a nested case-control study on 180 Mozambican pregnant women with placental malaria infection, and 180 controls within an intervention trial of malaria prevention. We genotyped 880 SNPs in a set of 64 functionally related genes involved in glycosylation and innate immunity. A SNP located in the gene FUT9, rs3811070, was significantly associated with placental malaria infection (OR = 2.31, permutation p-value = 0.028). Haplotypic analysis revealed a similarly strong association of a common haplotype of four SNPs including rs3811070. FUT9 codes for a fucosyl-transferase that is catalyzing the last step in the biosynthesis of the Lewis-x antigen, which forms part of the Lewis blood group-related antigens. These results therefore suggest an involvement of this antigen in the pathogenesis of placental malaria infection.

A targeted association study of immunity genes and networks suggests novel associations with placental malaria infection

A large proportion of the death toll associated with malaria is a consequence of malaria infection during pregnancy, causing up to 200,000 infant deaths annually. We previously published the first extensive genetic association study of placental malaria infection, and here we extend this analysis considerably, investigating genetic variation in over 9,000 SNPs in more than 1,000 genes involved in immunity and inflammation for their involvement in susceptibility to placental malaria infection. We applied a new approach incorporating results from both single gene analysis as well as gene-gene interactionson a protein-protein interaction network. We found suggestive associations of variants in the gene KLRK1 in the single geneanalysis, as well as evidence for associations of multiple members of the IL-7/IL-7R signalling cascade in the combined analysis. To our knowledge, this is the first large-scale genetic study on placental malaria infection to date, opening the door for follow-up studies trying to elucidate the genetic basis of this neglected form of malaria.

Fighting against Malaria: Prevent wars while waiting for the "miraculous" vaccine

The World Health Organization estimates that 300 million clinical cases of malaria occur annually and observed that during the 80's and part of the 90's its incidence increased. In this paper we explore the influence of refugees from civil wars on the incidence of malaria in the refugee-receiving countries. Using civil wars as an instrumental variable we show that for each 1,000 refugees there are between 2,000 and 2,700 cases of malaria in the refugee receiving country. On average 13% of the cases of malaria reported by the WHO are caused by forced migration as a consequence of civil wars.

Ultrastructural data on the life cycle of the parasite, Perkinsus atlanticus (Apicomplexa), on the clam, Ruditapes phillipinarum, in the Mediterranean

An Apicomplexan Perkinsus species has been found parasitizing the clam Ruditapes philippinarum (= Tapes semidecussatus) collected on the Mediterranean coast in the region of the Ebro Delta (Tarragona, Spain). Light and transmission electron microscopy were used to study different stages of this parasite during zoosporulation induced by incubation in thioglycollate medium and seawater. During incubation the trophozoites began zoosporulation, which originated prezoosporangia and zoosporangia at different developmental stages. Successive cytokinesis and nucleokinesis gave rise to prezoospores, which became elongate and differentiated in biflagellated zoospores. The latter presented large mitochondria and an apical complex formed by a conoid, polar ring, micronemes, rhophtries and subpellicular microtubules. The zoosporangium wall showed some typical lamosomes and a discharge tube developed in early phases of incubation. Ultrastructural data were compared with the only four species of the genus Perkinsus previously described. The morphological data, the host and the geographic proximity suggest that the species located on the Mediterranean coast was Perkinsus atlanticus.

Spermiogenesis and spermatozoon ultrastructure of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Gadiformes: Merlucciidae)

Spermiogenesis and the ultrastructure of the spermatozoon of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Linnaeus, 1758), have been studied by means of transmission electron microscopy. Spermiogenesis involves firstly the formation of a differentiation zone. It is characterized by the presence of two centrioles associated with striated rootlets, an intercentriolar body and an electron-dense material in the apical region of this zone. Later, two flagella develop from the centrioles, growing orthogonally in relation to the median cytoplasmic process. Flagella then undergo a rotation of 90° until they become parallel to the median cytoplasmic process, followed by the proximodistal fusion of the flagella with the median cytoplasmic process. The nucleus elongates and afterwards it migrates along the spermatid body. Spermiogenesis finishes with the appearance of the apical cone surrounded by the single helical crested body at the base of the spermatid. Finally, the narrowing of the ring of arched membranes detaches the fully formed spermatozoon. The mature spermatozoon of C. crassiceps is filiform and contains two axonemes of the 9 + '1' trepaxonematan pattern, a parallel nucleus, parallel cortical microtubules, and electron-dense granules of glycogen. The anterior extremity of the gamete exhibits a short electron-dense apical cone and one crested body, which turns once around the sperm cell. The first axoneme is surrounded by a ring of thick cortical microtubules that persist until the appearance of the second axoneme. Later, these thick cortical microtubules disappear and thus, the mature spermatozoon exhibits two bundles of thin cortical microtubules. The posterior extremity of the male gamete presents only the nucleus. Results are discussed and compared particularly with the available ultrastructural data on the former 'pseudophyllideans'. Two differences can be established between spermatozoa of Bothriocephalidea and Diphyllobothriidea, the type of spermatozoon (II vs I) and the presence/absence of the ring of cortical microtubules.

Spermiogenesis and spermatozoon ultrastructure of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Gadiformes: Merlucciidae)

Spermiogenesis and the ultrastructure of the spermatozoon of the bothriocephalidean cestode Clestobothrium crassiceps (Rudolphi, 1819), a parasite of the teleost fish Merluccius merluccius (Linnaeus, 1758), have been studied by means of transmission electron microscopy. Spermiogenesis involves firstly the formation of a differentiation zone. It is characterized by the presence of two centrioles associated with striated rootlets, an intercentriolar body and an electron-dense material in the apical region of this zone. Later, two flagella develop from the centrioles, growing orthogonally in relation to the median cytoplasmic process. Flagella then undergo a rotation of 90° until they become parallel to the median cytoplasmic process, followed by the proximodistal fusion of the flagella with the median cytoplasmic process. The nucleus elongates and afterwards it migrates along the spermatid body. Spermiogenesis finishes with the appearance of the apical cone surrounded by the single helical crested body at the base of the spermatid. Finally, the narrowing of the ring of arched membranes detaches the fully formed spermatozoon. The mature spermatozoon of C. crassiceps is filiform and contains two axonemes of the 9 + '1' trepaxonematan pattern, a parallel nucleus, parallel cortical microtubules, and electron-dense granules of glycogen. The anterior extremity of the gamete exhibits a short electron-dense apical cone and one crested body, which turns once around the sperm cell. The first axoneme is surrounded by a ring of thick cortical microtubules that persist until the appearance of the second axoneme. Later, these thick cortical microtubules disappear and thus, the mature spermatozoon exhibits two bundles of thin cortical microtubules. The posterior extremity of the male gamete presents only the nucleus. Results are discussed and compared particularly with the available ultrastructural data on the former 'pseudophyllideans'. Two differences can be established between spermatozoa of Bothriocephalidea and Diphyllobothriidea, the type of spermatozoon (II vs I) and the presence/absence of the ring of cortical microtubules.

Ultrastructure of vitellogenesis and vitellocytes in the trypanorhynch cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax

This is the first TEM examination of vitellogenesis in the cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax and a member of a little-studied trypanorhynch family, the Aporhynchidae. The synthetic activity of vitellocytes plays two important functions in the developmental biology of cestodes: (1) their shell-globules serve in eggshell formation; and (2) their accumulated reserves of glycogen and lipids represent a food source for the developing embryo. In A. menezesi, vitelline follicles consist of cells at various stages of development, from peripheral, immature cells of the gonial type to mature cells towards the centre of the follicle. These stages are: (I) immature; (II) early differentiation; (III) advanced maturation; and (IV) mature. Gradual changes involved in this process occur within each stage. Vitellogenesis involves: (1) an increase in cell volume; (2) the development of a smooth endoplasmic reticulum and an accelerated formation and accumulation of both unsaturated and saturated lipid droplets, along with their continuous enlargement and fusion; (3) the formation of individual β-glycogen particles and their accumulation in the form of glycogen islands scattered among lipid droplets in the cytoplasm of maturing and mature vitellocytes; (4) the rapid accumulation of large, moderately saturated lipid droplets accompanied by dense accumulations of β-glycogen along with proteinaceous shell-globules or shell-globule clusters in the peripheral layer during the advanced stage of maturation; (5) the development of cisternae of granular endoplasmic reticulum that produce dense, proteinaceous shell-globules; (6) the development of Golgi complexes engaged in the packaging of this material; and (7) the progressive and continuous enlargement of shell-globules into very large clusters in the peripheral layer during the advanced stage of maturation. Vitellogenesis in A. menezesi, only to some extent, resembles that previously described for four other trypanorhynchs. It differs in: (i) the reversed order of secretory activities in the differentiating vitellocytes, namely the accumulation of large lipid droplets accompanied by glycogenesis or β-glycogen formation during early differentiation (stage II), i.e. before the secretory activity, which is predominantly protein synthesis for shell-globule formation (stage III); (ii) the very heavy accumulation of large lipid droplets during the final stage of cytodifferentiation (stage IV); and (iii) the small number of β-glycogen particles present in mature vitellocytes. Ultracytochemical staining with PA-TCH-SP for glycogen proved positive for a small number of β-glycogen particles in differentiating and mature vitellocytes. Hypotheses, concerning the interrelationships of patterns of vitellogenesis, possible modes of egg formation, embryonic development and life-cycles, are commented upon.

Ultrastructure of vitellogenesis and vitellocytes in the trypanorhynch cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax

This is the first TEM examination of vitellogenesis in the cestode Aporhynchus menezesi, a parasite of the velvet belly lanternshark Etmopterus spinax and a member of a little-studied trypanorhynch family, the Aporhynchidae. The synthetic activity of vitellocytes plays two important functions in the developmental biology of cestodes: (1) their shell-globules serve in eggshell formation; and (2) their accumulated reserves of glycogen and lipids represent a food source for the developing embryo. In A. menezesi, vitelline follicles consist of cells at various stages of development, from peripheral, immature cells of the gonial type to mature cells towards the centre of the follicle. These stages are: (I) immature; (II) early differentiation; (III) advanced maturation; and (IV) mature. Gradual changes involved in this process occur within each stage. Vitellogenesis involves: (1) an increase in cell volume; (2) the development of a smooth endoplasmic reticulum and an accelerated formation and accumulation of both unsaturated and saturated lipid droplets, along with their continuous enlargement and fusion; (3) the formation of individual β-glycogen particles and their accumulation in the form of glycogen islands scattered among lipid droplets in the cytoplasm of maturing and mature vitellocytes; (4) the rapid accumulation of large, moderately saturated lipid droplets accompanied by dense accumulations of β-glycogen along with proteinaceous shell-globules or shell-globule clusters in the peripheral layer during the advanced stage of maturation; (5) the development of cisternae of granular endoplasmic reticulum that produce dense, proteinaceous shell-globules; (6) the development of Golgi complexes engaged in the packaging of this material; and (7) the progressive and continuous enlargement of shell-globules into very large clusters in the peripheral layer during the advanced stage of maturation. Vitellogenesis in A. menezesi, only to some extent, resembles that previously described for four other trypanorhynchs. It differs in: (i) the reversed order of secretory activities in the differentiating vitellocytes, namely the accumulation of large lipid droplets accompanied by glycogenesis or β-glycogen formation during early differentiation (stage II), i.e. before the secretory activity, which is predominantly protein synthesis for shell-globule formation (stage III); (ii) the very heavy accumulation of large lipid droplets during the final stage of cytodifferentiation (stage IV); and (iii) the small number of β-glycogen particles present in mature vitellocytes. Ultracytochemical staining with PA-TCH-SP for glycogen proved positive for a small number of β-glycogen particles in differentiating and mature vitellocytes. Hypotheses, concerning the interrelationships of patterns of vitellogenesis, possible modes of egg formation, embryonic development and life-cycles, are commented upon.

Ultrastructure of the spermatozoon of the digenean Lecithocladium excisum (Rudolphi, 1819) (Hemiuroidea: Hemiuridae), a parasite of marine teleosts in Senegal

The present study describes the ultrastructure of the mature spermatozoon of Lecithocladium excisum (Rudolphi, 1819) (Digenea: Hemiuroidea: Hemiuridae) from the stomach of the marine teleost Scomber japonicus Houttuyn (Scombridae) captured in the Atlantic Ocean, off Dakar (Senegal). The ultrastructural organization of the spermatozoon of L. excisum follows the general model described in most digeneans. It presents two axonemes of the 9+'1' pattern of the Trepaxonemata, nucleus, mitochondrion and parallel cortical microtubules, among other characters. However, some particularities of the spermatozoon of L. excisum are (i) the presence of a membranous ornamentation not associated with cortical microtubules in its anterior extremity, (ii) the presence of a very reduced number of cortical microtubules located only in the ventral side of the spermatozoon and (iii) the absence of several structures described in most digeneans such as spine-like bodies and cytoplasmic expansions.

Spermiogenesis and spermatozoon of the liver fluke Mediogonimus jourdanei (Microphalloidea: Prosthogonimidae), a parasite of Myodes glareolus (Rodentia: Cricetidae)

Spermatological characters of the liver fluke Mediogonimus jourdanei Mas-Coma et Rocamora, 1978 were studied by means of transmission and scanning electron microscopy. Spermiogenesis begins with the formation of the differentiation zone containing two centrioles associated with striated rootlets and an intercentriolar body. These two centrioles originate two free flagella that undergo a 90 degrees rotation before fusing with the median cytoplasmic process. Both nuclear and mitochondrial migrations toward the median cytoplasmic process occur before the proximodistal fusion of flagella. Finally, the constriction of the ring of arched membranes gives rise to the young spermatozoon. The mature sperm of M. jourdanei measures about 260 microm and presents two axonemes of different lengths with the typical pattern of the Trepaxonemata, two bundles of parallel cortical microtubules, one mitochondrion, a nucleus and granules of glycogen. An analysis of all the microphalloidean species studied to date emphasised some differences in certain characters found in Maritrema linguilla Jägerskiöld, 1908 and Ganeo tigrinum Mehra et Negi, 1928 in comparison to those in the remaining microphalloideans. The presence and variability of such ultrastructural characters according to family, superfamily or order have led several authors to propose their use in the analysis of trematode relationships and phylogeny. Therefore, apart from producing new data on the family Prosthogonimidae, the present study also compares the spermatological organization of M jourdanei with other available ultrastructural studies focusing on the Microphalloidea.

Ultrastructure of the spermatozoon of the digenean Lecithocladium excisum (Rudolphi, 1819) (Hemiuroidea: Hemiuridae), a parasite of marine teleosts in Senegal

The present study describes the ultrastructure of the mature spermatozoon of Lecithocladium excisum (Rudolphi, 1819) (Digenea: Hemiuroidea: Hemiuridae) from the stomach of the marine teleost Scomber japonicus Houttuyn (Scombridae) captured in the Atlantic Ocean, off Dakar (Senegal). The ultrastructural organization of the spermatozoon of L. excisum follows the general model described in most digeneans. It presents two axonemes of the 9+'1' pattern of the Trepaxonemata, nucleus, mitochondrion and parallel cortical microtubules, among other characters. However, some particularities of the spermatozoon of L. excisum are (i) the presence of a membranous ornamentation not associated with cortical microtubules in its anterior extremity, (ii) the presence of a very reduced number of cortical microtubules located only in the ventral side of the spermatozoon and (iii) the absence of several structures described in most digeneans such as spine-like bodies and cytoplasmic expansions.

Spermiogenesis and spermatozoon of the liver fluke Mediogonimus jourdanei (Microphalloidea: Prosthogonimidae), a parasite of Myodes glareolus (Rodentia: Cricetidae)

Spermatological characters of the liver fluke Mediogonimus jourdanei Mas-Coma et Rocamora, 1978 were studied by means of transmission and scanning electron microscopy. Spermiogenesis begins with the formation of the differentiation zone containing two centrioles associated with striated rootlets and an intercentriolar body. These two centrioles originate two free flagella that undergo a 90 degrees rotation before fusing with the median cytoplasmic process. Both nuclear and mitochondrial migrations toward the median cytoplasmic process occur before the proximodistal fusion of flagella. Finally, the constriction of the ring of arched membranes gives rise to the young spermatozoon. The mature sperm of M. jourdanei measures about 260 microm and presents two axonemes of different lengths with the typical pattern of the Trepaxonemata, two bundles of parallel cortical microtubules, one mitochondrion, a nucleus and granules of glycogen. An analysis of all the microphalloidean species studied to date emphasised some differences in certain characters found in Maritrema linguilla Jägerskiöld, 1908 and Ganeo tigrinum Mehra et Negi, 1928 in comparison to those in the remaining microphalloideans. The presence and variability of such ultrastructural characters according to family, superfamily or order have led several authors to propose their use in the analysis of trematode relationships and phylogeny. Therefore, apart from producing new data on the family Prosthogonimidae, the present study also compares the spermatological organization of M jourdanei with other available ultrastructural studies focusing on the Microphalloidea.

Ultrastructural study of spermiogenesis and the spermatozoon of the proteocephalidean cestode Barsonella lafoni de Chambrier et al., 2009, a parasite of the catfish Clarias gariepinus (Burchell, 1822) (Siluriformes, Clariidae)

Spermiogenesis in the proteocephalidean cestode Barsonella lafoni de Chambrier et al., 2009 shows typical characteristics of the type I spermiogenesis. These include the formation of distal cytoplasmic protrusions forming the differentiation zones, lined by cortical microtubules and containing two centrioles. An electron-dense material is present in the apical region of the differentiation zone during the early stages of spermiogenesis. Each centriole is associated to a striated rootlet, being separated by an intercentriolar body. Two free and unequal flagella originate from the centrioles and develop on the lateral sides of the differentiation zone. A median cytoplasmic process is formed between the flagella. Later these flagella rotate, become parallel to the median cytoplasmic process and finally fuse proximodistally with the latter. It is interesting to note that both flagellar growth and rotation are asynchronous. Later, the nucleus enlarges and penetrates into the spermatid body. Finally, the ring of arching membranes is strangled and the young spermatozoon is detached from the residual cytoplasm. The mature spermatozoon presents two axonemes of the 9 +"1" trepaxonematan pattern, crested body, parallel nucleus and cortical microtubules, and glycogen granules. Thus, it corresponds to the type II spermatozoon, described in almost all Proteocephalidea. The anterior extremity of the gamete is characterized by the presence of an apical cone surrounded by the lateral projections of the crested body. An arc formed by some thick and parallel cortical microtubules appears at the level of the centriole. They surround the centriole and later the first axoneme. This arc of electron-dense microtubules disorganizes when the second axoneme appears, and then two parallel rows of thin cortical microtubules are observed. The posterior extremity of the male gamete exhibits some cortical microtubules. This type of posterior extremity has never been described in proteocephalidean cestodes. The ultrastructural features of the spermatozoon/spermiogenesis of the Proteocephalidea species are analyzed and compared.