Ryuichiro Maeda

Isolation of Neospora caninum from the brain of a pregnant sheep.

Neospora caninum was isolated from the brain of a naturally infected pregnant sheep by inoculation of immunodeficient mice with a homogenate of the brain tissue. The ewe showed no clinical signs. Tachyzoites were observed in the tissues of the nu/nu mice injected with the brain tissue homogenate and the diagnosis was confirmed by immunohistochemical staining with anti-N. caninum antibodies and by detecting N. caninum-specific DNA by polymerase chain reaction.

Interactive transcriptome analysis of malaria patients and infecting Plasmodium falciparum

To understand the molecular mechanisms of parasitism in vivo, it is essential to elucidate how the transcriptomes of the human hosts and the infecting parasites affect one another. Here we report the RNA-seq analysis of 116 Indonesian patients infected with the malaria parasite Plasmodium falciparum (Pf). We extracted RNAs from their peripheral blood as a mixture of host and parasite transcripts and mapped the RNA-seq tags to the human and Pf reference genomes to separate the respective tags. We were thus able to simultaneously analyze expression patterns in both humans and parasites. We identified human and parasite genes and pathways that correlated with various clinical data, which may serve as primary targets for drug developments. Of particular importance, we revealed characteristic expression changes in the human innate immune response pathway genes including TLR2 and TICAM2 that correlated with the severity of the malaria infection. We also found a group of transcription regulatory factors, JUND, for example, and signaling molecules, TNFAIP3, for example, that were strongly correlated in the expression patterns of humans and parasites. We also identified several genetic variations in important anti-malaria drug resistance-related genes. Furthermore, we identified the genetic variations which are potentially associated with severe malaria symptoms both in humans and parasites. The newly generated data should collectively lay a unique foundation for understanding variable behaviors of the field malaria parasites, which are far more complex than those observed under laboratory conditions.

A 14-3-3 protein homologue is expressed in feline enteroepithelial-stages of Toxoplasma gondii

Fourteen cDNA clones encoding epitopes of proteins of Toxoplasma gondii feline enteroepithelial-stages parasites were isolated and expressed in Escherichia coli in an effort to determine the antigenecity of the parasites. Sequence analysis showed that four of the cDNA clones had a 930-bp open-reading frame encoding a product showing similarity to the 14-3-3 protein mRNA sequence.(1) Southern hybridization of DIG-labeled positive clone with T. gondii genomic DNA cleaved with EcoRI, BamHI and HindIII resulted in one or two bands in each case. In an immunofluorescence assay, polyclonal and monoclonal antibodies raised against the expressed protein showed strong reactivity with feline enteroepithelial-stages parasites and sporozoites. In a complementation assay in which a plasmid carrying the protein-coding region of the isolated cDNA was introduced into a Saccharomyces cerevisiae mutant, strain DS9-22, the expressed protein showed complementation of the function of the 14-3-3 protein in yeast transformants. These findings suggest that T. gondii parasites produce a protein showing partial homology with members of the 14-3-3 protein family and this protein is expressed in feline enteroepithelial-stages parasites.

Computed tomography (CT) observation of pulmonary emboli caused by long-term administration of ivermectin in dogs experimentally infected with heartworms

Some studies have reported the adulticidal effect of long-term ivermectin (IVM) administration on adult heartworms in canines; however, there are no detailed reports on the course of the pulmonary artery embolism caused by the bodies of dead heartworms during the administration period. In this study, the pulmonary embolism caused over time by the dead worms was observed using computed tomography (CT). We subcutaneously inoculated 2 beagles with 100 infective third-stage larvae (L3) of Dirofilaria immitis. The dogs were orally administered a formulation containing 272 microg of IVM and 652 mg of pyrantel pamoate (Panamectin Chewables P272; Meiji Seika, Tokyo, Japan) at monthly intervals, beginning from 10 months after the subcutaneous inoculation. Along with IVM administration, periodic CT examination of the chest was performed. At 15 months after the initiation of IVM administration, the dogs were euthanized, the living heartworms were collected, and histopathological examination was performed. Starting from 1 month after the IVM administration, peripheral dilation of the pulmonary artery (suspected to be pulmonary embolism) and pneumonia were observed in the CT images; however, these findings improved over time. The appearance and disappearance of these lesions were observed in all the lobes during the IVM administration period. During this period, the clinical symptoms of pulmonary embolism were not recognized. After 1 month of IVM administration, chest radiographic examination revealed radiopaque lesions in 1 dog. Only some of the lesions detected by CT could be detected by radiography. Using echocardiography, heartworms were observed in the pulmonary arteries of both dogs from 6 months after subcutaneous inoculation to the end of the study period. Microfilaria disappeared from the peripheral blood at 1 month after IVM administration in 1 dog, and at 7 months in the other dog. The adult heartworm antigen test yielded positive results starting from 6 months after subcutaneous inoculation in 1 dog and after 7 months in the other dog; these results remained positive until the end of the study period. After the initiation of IVM administration, the ALP and CK levels were transiently elevated. The number of surviving adult worms collected at necropsy was 25 in 1 dog and 31 in the other. Histopathological examination revealed that the peripheral pulmonary artery dilation detected by CT was the embolus that resulted from the bodies of the dead heartworms. Moreover, vessel recanalization and inflammation along with lymphocyte infiltration around the vessels was observed. These results revealed that long-term IVM administration has a gradual adulticidal effect on heartworms in canines and embolism. From recovery findings showed pulmonary embolism in the CT image and histopathologic examination, long-term IVM administration can potentially be used for adulticidal treatment in clinical cases where it is difficult to perform surgical extirpation and administer arsenic therapy.

Quantitative analysis of microfilarial periodicity of Dirofilaria immitis in cats

Microfilarial periodicity of Dirofilaria immitis in the venous blood of infected cats was analyzed by a trigonometric model. Cats were infected by subcutaneous transplantation with 120-day-old juvenile D. immitis. Microfilariae in the blood were first observed 98 days after transplantation. Blood was collected at 4h intervals for a 24h period, and examinations were repeated five times in two cats. The calculated periodicity index was 75.1 and 50.3 in these two cats. The estimated hour of peak microfilarial density ranged from 1.00 to 2.84h. Thus, the periodicity of microfilariae of D. immitis in the blood of cats was characterized as nocturnally sub-periodic.

Prevalence of Toxoplasma gondii and Neospora caninum in Sika Deer from Eastern Hokkaido, Japan

Brain and serum were collected from 120 and 12 free-ranging sika deer (Cervus nippon yesoensis), respectively, from six regions in eastern Hokkaido during controlled hunts in the autumn of 2003. Brains were tested for Neospora caninum and Toxoplasma gondii DNA by polymerase chain reaction (PCR) assays. Antibodies to Toxoplasma gondii were measured by means of a latex agglutination test. No brain tested positive for either type of DNA, and no antibody to Toxoplasma gondii was detected in serum, suggesting a low prevalence of infection with these organisms in free-ranging sika deer from eastern Hokkaido. Further examination of multiple tissues by PCR and serologic surveys will be necessary to confirm this.

Toxoplasma gondii does not persist in goldfish (Carassius auratus).

Recent reports of toxoplasmosis in marine mammals raise concern that cold-blooded marine animals are a potential source of Toxoplasma gondii infection. To examine the transmissibility of T. gondii to fish, we observed the development of T. gondii tachyzoites inoculated into oviduct epithelial cells of goldfish (Carassius auratus) microscopically in vitro. Further, the survival period of tachyzoites inoculated into goldfish muscle was bioassayed in mice and through PCR analysis. In cell cultures at 37 C, both RH and Beverley strains of T. gondii tachyzoites had penetrated into cells at 6 hr post inoculation, and were multiplying. In cell cultures at 33 C, many tachyzoites of both strains attached to the host cells, but no intracellular tachyzoites were observed at 24 hr post inoculation. In the T. gondii inoculated goldfish kept at 33 C, tachyzoite DNA was detected in the inoculated region on day 3, but not on day 7. When inoculated goldfish were kept at 37 C, live tachyzoites were seen at the inoculation site on day 3, but not on day 7. These results suggest that T. gondii does not persist in fish.

Development of Neospora caninum Cultured with Human Serum In Vitro and In Vivo

Because there has been no report of symptomatic Neospora caninum infection in humans, we examined the effect of human serum on the parasites growth in either a bovine angioendothelial cell or Caco-2 cell culture in vitro and in immunocompromised mice in vivo. There was no difference in intracellular parasite numbers between cells incubated with human serum at 24 hr after challenge and those incubated with fetal bovine serum (FBS), which has no titer for the anti–N. caninum agglutination antibody test. Serum of sheep infected with N. caninum, which has the anti–N. caninum antibody, reduced the numbers of the intracellular parasite significantly. We also showed that there was no inhibitory effect on the intracellular multiplication of the parasite in cells incubated with human serum through incorporation of 3H-uracil. CB-17 scid mice administered human serum daily and challenged with N. caninum died on day 20 or 22 after challenge, when large numbers of parasite clusters were found in the brain, oviduct, adrenal gland, lung, stomach, spleen, skeletal muscle, pancreas, and mesenteric lymph nodes. Scid mice administered FBS survived until the end of the experiment. These results suggest that adult human serum may have no inhibitory effect on the development of N. caninum in vitro and in vivo.

Relationship between liver disorders and protection against Eimeria stiedai infection in rabbits immunized with soluble antigens from the bile of infected rabbits

Soluble antigens exist in the bile of rabbits infected with Eimeria stiedai (E. stiedai) in the acute phase, and rabbits immunized with the antigens show resistance against the infection. In this study, the liver function of rabbits immunized either with the soluble antigens or PBS were examined following the parasite challenge. Rabbits immunized with PBS shed a number of oocysts and showed an increase in r-glutamyltransferase (GGT) activity and a decrease in blood Indocyanine green (ICG) clearance. However, rabbits immunized with the soluble antigens shed a lower number of oocysts and showed a transient increase of alanine-aminotransferase (ALT) activity on Day 8 post-challenge (p.c.). The blood Indocyanine green clearance of the rabbits showed no change throughout the experiment. By histopathological observation of the liver, a number of merozoites were found in the biliary ducts on Day 8 post-challenge in the non-immunized rabbits. In contrast, a number of lymphocytes and neutrophilic leukocytes assembled around the biliary ducts of the immunized rabbits, but few parasites were found there on Day 8 post-challenge. These results suggest that the soluble antigens stimulate local immune reactions, for example around the biliary ducts, resulting in elimination of the parasites development.

A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer

BackgroundA simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer.MethodsWe generated specific LAMP primers targeting the 18S–rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species.ResultsOur LAMP method allowed amplification of all targeted 18S–rRNA genes of the reference plasmids with detection limits of 10–100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR.ConclusionsOur diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.