Hajime Honma

Plasmodium cynomolgi genome sequences provide insight into Plasmodium vivax and the monkey malaria clade

P. cynomolgi, a malaria-causing parasite of Asian Old World monkeys, is the sister taxon of P. vivax, the most prevalent malaria-causing species in humans outside of Africa. Because P. cynomolgi shares many phenotypic, biological and genetic characteristics with P. vivax, we generated draft genome sequences for three P. cynomolgi strains and performed genomic analysis comparing them with the P. vivax genome, as well as with the genome of a third previously sequenced simian parasite, Plasmodium knowlesi. Here, we show that genomes of the monkey malaria clade can be characterized by copy-number variants (CNVs) in multigene families involved in evasion of the human immune system and invasion of host erythrocytes. We identify genome-wide SNPs, microsatellites and CNVs in the P. cynomolgi genome, providing a map of genetic variation that can be used to map parasite traits and study parasite populations. The sequencing of the P. cynomolgi genome is a critical step in developing a model system for P. vivax research and in counteracting the neglect of P. vivax.

Cryptosporidium genotypes and subtypes in dairy calves in Egypt

Neonatal calves are prone to Cryptosporidium infection resulting in economic loss as well as a significant source for zoonotic infection. This study was devoted to ascertain the prevalence and molecular analysis of Cryptosporidium in dairy calves at Kafr El Sheikh Province, Egypt. Twenty-nine out of 96 faecal specimens collected from calves, less than 6 weeks of ages, microscopically showed cryptosporidia oocysts (prevalence 30.2%). Among 29 positives, 26 samples were clearly sequenced for the SSU rDNA gene and the Cryptosporidium oocyst wall protein gene (COWP). Homology search revealed that 2 samples were C. andersoni and 24 isolates were C. parvum genotype II. By sequence analysis of the high polymorphic 60-kDa glycoprotein gene, 23 samples of C. parvum belonged to the allele IId (subtypes IIdA20G1), and one sample belonged to the allele IIa (subtype IIaA15G2R1). Prevailing of allele family IId is a unique observation, contrasting the conception that IIa has been the most prevailing allele in calves and cattle generally in other countries such as in Europe and the USA. To our knowledge, this is the first molecular report about genotyping and subtyping of Cryptosporidium in Egypt.

Concatenated mitochondrial DNA of the coccidian parasite Eimeria tenella.

Apicomplexan parasites of the genus Plasmodium, pathogens causing malaria, and the genera Babesia and Theileria, aetiological agents of piroplasmosis, are closely related. However, their mitochondrial (mt) genome structures are highly divergent: Plasmodium has a concatemer of 6-kb unit and Babesia/Theileria a monomer of 6.6- to 8.2-kb with terminal inverted repeats. Fragmentation of ribosomal RNA (rRNA) genes and gene arrangements are remarkably distinctive. To elucidate the evolutionary origin of this structural divergence, we determined the mt genome of Eimeria tenella, pathogens of coccidiosis in domestic fowls. Analysis revealed that E. tenella mt genome was concatemeric with similar protein-coding genes and rRNA gene fragments to Plasmodium. Copy number was 50-fold of the nuclear genome. Evolution of structural divergence in the apicomplexan mt genomes is discussed.

The first detection of Cryptosporidium deer-like genotype in cattle in Japan

The general perception is that cattle are major reservoirs for Cryptosporidium parvum infections in humans and that C. parvum is a major cause of diarrhea and production loss in cattle. Adult cattle may play an important role as cryptic carrier of the infection. Cryptosporidium spp. in asymptomatic adult dairy cattle from some farms around Osaki area, Miyagi prefecture, Japan, was examined on a field visit during August, 2007, by polymerase chain reaction techniques for detection, genotyping, and subtyping. Cryptosporidium oocysts were detected in the feces of five out of 50 animals. Of the five Cryptosporidium-positive specimens available for molecular analysis, C. parvum was identified in three specimens, Cryptosporidium deer-like genotype in one, and Cryptosporidium andersoni in one specimen. Amplification of Cpgp60 from C. andersoni and Cryptosporidium deer-like genotype samples revealed that these samples have light concurrent C. parvum infection. Sequence analysis of the 60-kDa glycoprotein gene indicated that all C. parvum samples are IIa subtype. Detection of Cryptosporidium deer-like genotype is geographically unique in Japan. The genetic diversity of Cryptosporidium in dairy cattle in Japan may be much greater than that reported before.

Geographic differentiation of polymorphism in the Plasmodium falciparum malaria vaccine candidate gene SERA5.

SERA5 is regarded as a promising malaria vaccine candidate of the most virulent human malaria parasite Plasmodium falciparum. SERA5 is a 120 kDa abundantly expressed blood-stage protein containing a papain-like protease. Since substantial polymorphism in blood-stage vaccine candidates may potentially limit their efficacy, it is imperative to fully investigate polymorphism of the SERA5 gene (sera5). In this study, we performed evolutionary and population genetic analysis of sera5. The level of inter-species divergence (kS=0.076) between P. falciparum and Plasmodium reichenowi, a closely related chimpanzee malaria parasite is comparable to that of housekeeping protein genes. A signature of purifying selection was detected in the proenzyme and enzyme domains. Analysis of 445 near full-length P. falciparum sera5 sequences from nine countries in Africa, Southeast Asia, Oceania and South America revealed extensive variations in the number of octamer repeat (OR) and serine repeat (SR) regions as well as substantial level of single nucleotide polymorphism (SNP) in non-repeat regions (2562 bp). Remarkably, a 14 amino acid sequence of SERA5 (amino acids 59-72) that is known to be the in vitro target of parasite growth inhibitory antibodies was found to be perfectly conserved in all 445 worldwide isolates of P. falciparum evaluated. Unlike other major vaccine target antigen genes such as merozoite surface protein-1, apical membrane antigen-1 or circumsporozoite protein, no strong evidence for positive selection was detected for SNPs in the non-repeat regions of sera5. A biased geographical distribution was observed in SNPs as well as in the haplotypes of the sera5 OR and SR regions. In Africa, OR- and SR-haplotypes with low frequency (<5%) and SNPs with minor allele frequency (<5%) were abundant and were mostly continent-specific. Consistently, significant genetic differentiation, assessed by the Wrights fixation index (Fst) of inter-population variance in allele frequencies, was detected for SNPs and both OR- and SR-haplotypes among almost all parasite populations. The exception was parasite populations between Tanzania and Ghana, suggesting frequent gene flow in Africa. The present study points to the importance of investigating whether biased geographical distribution for SNPs and repeat variants in the OR and SR regions affect the reactivity of human serum antibodies to variants.

Prevalence and risk factors associated with Cryptosporidium oocysts shedding in pigs in Central Vietnam

We investigated the prevalence and risk factors associated with Cryptosporidium oocysts shedding in pigs in Central Vietnam. A total of 740 single fecal samples collected from diarrheic and non-diarrheic pigs on 89 farms were screened by the modified Ziehl-Neelsen staining method. Prevalence at the animal and the farm levels were 18.1% (134/740) and 71.9% (64/89), respectively. Risk factors for the infection were identified using univariate and multivariate logistic regression analysis. The results revealed that age, sanitary condition and topography were significantly associated with oocyst shedding (P<0.05). Pre-weaned piglets were at the highest risk for infection, followed by post-weaners, sows and finishing pigs. Good sanitary conditions showed positive effects in decreasing oocysts shedding. Topographically, Cryptosporidium was more common in mountainous zone than that in coastal delta zone. There was an association between the occurrence of diarrhea and the level of Cryptosporidium oocyst excretion within infected pigs. This is the first epidemiological investigation of prevalence and risk factors of Cryptosporidium in pigs in Vietnam.

Recent increase of genetic diversity in Plasmodium vivax population in the Republic of Korea

BackgroundThe reemergence of Plasmodium vivax in South Korea since 1993 represents a serious public health concern. Despite the importance in understanding genetic diversity for control strategies, however, studies remain inconclusive with the general premise that due to low rate of malaria transmission, there is generally low genetic diversity with very few strains involved. In this study, the genetic diversity and population structure of P. vivax in South Korea were explored by analysing microsatellite polymorphism.MethodsSequences for 13 microsatellite loci distributed across the twelve chromosomes of P. vivax were obtained from 58 South Korean isolates collected during two sampling periods, namely 1997-2000 and 2007. The sequences were used for the analysis of expected heterozygosity and multilocus genotype diversity. Population structure was evaluated using STRUCTURE version 2.3.2. Linkage disequilibrium was also analysed to investigate the extent of outbreeding in the P. vivax population.ResultsMean expected heterozygosity significantly increased from 0.382 in 1997-2000 to 0.545 in 2007 (P < 0.05). The number of multilocus genotypes was 7 and 27; and genotype diversity was statistically significant (P < 0.01) at 0.661 and 0.995 in 1997-2000 and 2007, respectively. Analysis by STRUCTURE showed a more complex population structure in 2007 than in 1997-2000. Linkage disequilibrium between 13 microsatellites, although significant in both time points, was notably lower in 2007.ConclusionsThe present microsatellite analysis clearly showed recent increase of genetic diversity and recent relaxation of the strong population structure observed in 1997-2000. These results suggest that multiple genotypes not present previously recently migrated into South Korea, accompanied by substantial outbreeding between different genotypes.

Plasmodium falciparum mitochondrial genetic diversity exhibits isolation-by-distance patterns supporting a sub-Saharan African origin.

The geographical distribution of single nucleotide polymorphism (SNP) in the mitochondrial genome of the human malaria parasite Plasmodium falciparum was investigated. We identified 88 SNPs in 516 isolates from seven parasite populations in Africa, Southeast Asia and Oceania. Analysis of the SNPs postulated a sub-Saharan African origin and recovered a strong negative correlation between within-population SNP diversity and geographic distance from the putative African origin over Southeast Asia and Oceania. These results are consistent with those previously obtained for nuclear genome-encoded housekeeping genes, indicating that the pattern of inheritance does not substantially affect the geographical distribution of SNPs.

Redescription of Triplumaria selenica Latteur et al., 1970 (Ciliophora, Entodiniomorphida) and its phylogenetic position based on the infraciliary bands and 18SSU rRNA gene sequence

Triplumaria selenicaLatteur, Tuffrau and Wespes, 1970 was redescribed from pyridinated silver carbonate-impregnated specimens. Triplumaria selenica has a slit of the vestibular opening extending posteriorly along the left side of the vestibulum. The wide C-shaped adoral polybrachykinety extends along the ventral side of the vestibular opening. The narrow perivestibular polybrachykinety extends laterally along the dorsal side of the vestibular opening from the right end of the adoral polybrachykinety and forms a loop extending posteriorly along the vestibular slit to join to the left end of the adoral polybrachykinety. The 18SSU rRNA gene of T. selenica as well as those of six other entodiniomorphid species, Raabena bella, Blepharocorys curvigula, Entodinium longinucleatum, Eudiplodinium rostratum, Metadinium medium, and Ostracodinium gracile was sequenced. The neighbor joining and maximum parsimony phylogenetic trees were constructed to discuss the evolution of entodiniomorphs. Our results will support and extend Wolskas hypothesis: the ancestral forms of blepharocorythids have evolved into ophryoscolecids and Cycloposthium species via the ancestor of Triplumaria.

Accurate analysis of prevalence of coccidiosis in individually identified wild cranes in inhabiting and migrating populations in Japan

Eimeria gruis and E. reichenowi cause coccidiosis, a major parasitic disease of cranes. By non-invasive molecular approaches, we investigated the prevalence and genetic characterization of pathogens in two Japanese crane habitats; one is Hokkaido inhabited by the endangered red-crowned crane, and the other is Izumi in Kyushu where populations that consist mainly of vulnerable hooded and white-naped cranes migrate in winter. The non-invasively collected faecal samples from each wintering population were first subjected to host genomic DNA-targeted analyses to determine the sample origin and avoid sample redundancy. Extremely high prevalence was observed in the Izumi populations (> 90%) compared with the Hokkaido population (18-30%) by examining 470 specimens by microscopy and PCR-based capillary electrophoresis (PCR-CE), using genetic markers in the second internal transcribed spacer (ITS2). Correspondence analysis of PCR-CE data revealed differences in community composition of coccidia between hooded and white-naped cranes. 18S rRNA and ITS2 sequences were determined from single oocysts excreted by red-crowned and hooded cranes. Phylogenetic analysis of 18S rRNA suggested that E. reichenowi was polyphyletic while E. gruis was monophyletic. Together with PCR-CE data, these results indicate different host specificity among the E. reichenowi type. Our data suggest that E. reichenowi comprises multiple species.