Publications by authors named "Fumiya Kawahara"

13 Publications

  • Page 1 of 1

Transitions in morphological forms and rapid development of the asexual schizonts of Eimeria tenella through serial passaging in chicks.

Infect Genet Evol 2019 11 5;75:103993. Epub 2019 Aug 5.

Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka 598-8531, Japan.

Attenuated strains of avian Eimeria parasites, generated by the selection of precocious lines through serial passaging in chicks, have been used widely as live vaccines. Detailed morphological transitions including their life cycle depending on the passages remain poorly understood. Here, we showed early development and acceleration of transitions in morphological forms of the asexual schizonts of E. tenella that had been attenuated for virulence by serial passaging. Our results may be helpful in understanding parasitism, facilitating further molecular analyses such as comparative genomic or transcriptomic tests.
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http://dx.doi.org/10.1016/j.meegid.2019.103993DOI Listing
November 2019

Native SAG1 in Toxoplasma gondii lysates is superior to recombinant SAG1 for serodiagnosis of T. gondii infections in chickens.

Parasitol Int 2019 Apr 7;69:114-120. Epub 2019 Jan 7.

The United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; Department of Veterinary Parasitological Diseases, Faculty of Applied Biological Science, 1-1 Yanagido, Gifu 501-1193, Japan; Center for Highly Advanced Integration of Nano and Life Sciences, Gifu University (G-CHAIN), 1-1 Yanagido, Gifu 501-1193, Japan. Electronic address:

Toxoplasma gondii can infect almost all mammals and birds, including chickens. The aim of this study was to identify an appropriate immunogenic antigen for serodiagnosis of T. gondii infections in chickens. We examined serum samples from chickens that were intravenously or intraperitoneally infected with 10-10 tachyzoites of T. gondii strains PLK, RH, CTG, ME49 or TgCatJpGi1/TaJ using enzyme-linked immunosorbent assays (ELISAs), latex agglutination tests (LATs) and western blotting. Regardless of parasite strain or infection dose and route, the commercial LAT was positive for almost all sera collected 1 week post-infection. However, at 2 weeks post-infection, LATs were negative in the same birds. ELISAs using the Escherichia coli-produced recombinant T. gondii antigens SAG1 and GRA7 showed strong signals at 1-2 weeks post infection, but thereafter diminished for the majority of serum samples. In contrast, western blotting against crude tachyzoite antigens showed a persistent band up to 4 weeks post-infection. Sera from these chickens reacted much more strongly with SAG1 from crude tachyzoite antigens than with recombinant SAG1. Even in experimentally-infected birds whose parasite burdens in tissue were undetectable, sera still reacted with native SAG1. We tested sera from free-range chickens on a small farm in Ghana, Africa, using western blotting and found that the serum of one bird reacted with a single band of approximately 27 kDa, the putative molecular weight of SAG1. Thus we conclude that native SAG1, but not E. coli-produced recombinant SAG1, is suitable for serodiagnosis of T. gondii infections in chickens.
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http://dx.doi.org/10.1016/j.parint.2019.01.001DOI Listing
April 2019

Novel Characteristics of Mitochondrial Electron Transport Chain from .

Genes (Basel) 2019 01 8;10(1). Epub 2019 Jan 8.

School of Tropical Medicine and Global Health, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan.

is an intracellular apicomplexan parasite, which infects cecal epithelial cells from chickens and causes hemorrhagic diarrhea and eventual death. We have previously reported the comparative RNA sequence analysis of the sporozoite stage between virulent and precocious strains and showed that the expression of several genes involved in mitochondrial electron transport chain (ETC), such as type II NADH dehydrogenase (NDH-2), complex II (succinate:quinone oxidoreductase), malate:quinone oxidoreductase (MQO), and glycerol-3-phosphate dehydrogenase (G3PDH), were upregulated in virulent strain. To study mitochondrial ETC in detail, we developed a reproducible method for preparation of mitochondria-rich fraction from sporozoites, which maintained high specific activities of dehydrogenases, such as NDH-2 followed by G3PDH, MQO, complex II, and dihydroorotate dehydrogenase (DHODH). Of particular importance, we showed that sporozoite mitochondria possess an intrinsic ability to perform fumarate respiration (via complex II) in addition to the classical oxygen respiration (via complexes III and IV). Further analysis by high-resolution clear native electrophoresis, activity staining, and nano-liquid chromatography tandem-mass spectrometry (nano-LC-MS/MS) provided evidence of a mitochondrial complex II-III-IV supercomplex. Our analysis suggests that complex II from has biochemical features distinct to known orthologues and is a potential target for the development of new anticoccidian drugs.
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http://dx.doi.org/10.3390/genes10010029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6356742PMC
January 2019

Experimental induction of necrotic enteritis in chickens by a netB-positive Japanese isolate of Clostridium perfringens.

J Vet Med Sci 2017 Feb 15;79(2):350-358. Epub 2016 Dec 15.

Nippon Institute for Biological Science, 9-2221-1 Shinmachi, Ome, Tokyo 198-0024, Japan.

Necrotic enteritis (NE) is one of the most important bacterial diseases in terms of economic losses. Clostridium perfringens necrotic enteritis toxin B, NetB, was recently proposed as a new key virulent factor for the development of NE. The goal of this work was to develop a necrotic enteritis model in chickens by using a Japanese isolate of C. perfringens. The Japanese isolate has been found to contain netB gene, which had the same nucleotide and deduced amino acid sequences as those of prototype gene characterized in Australian strain EHE-NE18, and also expressed in vitro a 33-kDa protein identified as NetB toxin by nano-scale liquid chromatographic tandem mass spectrometry. In the challenge experiment, broiler chickens fed a commercial chicken starter diet for 14 days post-hatch were changed to a high protein feed mixed 50:50 with fishmeal for 6 days. At day 21 of age, feed was withheld for 24 hr, and each chicken was orally challenged twice daily with 2 ml each of C. perfringens culture (10 to 10 CFU) on 5 consecutive days. The gross necrotic lesions were observed in 90 and 12.5% of challenged and control chickens, respectively. To our knowledge, this is the first study that demonstrated that a netB-positive Japanese isolate of C. perfringens is able to induce the clinical signs and lesions characteristic of NE in the experimental model, which may be useful for evaluating the pathogenicity of field isolates, the efficacy of a vaccine or a specific drug against NE.
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http://dx.doi.org/10.1292/jvms.16-0500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326941PMC
February 2017

Transcriptional profiles of virulent and precocious strains of Eimeria tenella at sporozoite stage; novel biological insight into attenuated asexual development.

Infect Genet Evol 2016 06 23;40:54-62. Epub 2016 Feb 23.

Department of Parasitology, Kitasato University School of Medicine, 1-15-1 Kitasato, Minami-ku, Sagamihara, Kanagawa 252-0374, Japan. Electronic address:

Chicken coccidiosis is caused by Eimeria spp., particularly Eimeria tenella, and is characterized by watery or hemorrhagic diarrhea, resulting in death in severe cases. Precociously attenuated live vaccines are widely used to control the disease, and these are produced by serially passaging virulent strains through chickens, and the collection of oocysts from feces at progressively earlier time points during oocyst shedding. Sporozoites of the precocious strain rapidly enter the intestinal mucosa, and their subsequent asexual development reduces their growth. However, there have been few detailed genetic or transcriptional analyses of the strains. Here, we used RNA sequencing to gain novel biological insight into the pathogenicity and precocity of E. tenella. We compared the differential transcription in the sporozoites (the initial stage of endogenous development) of virulent and precocious strains by mapping the sequence reads onto the draft genome of E. tenella. About 90% of the reads from both strains were mapped to the genome, and 16,630 estimated transcript regions were identified. Using Gene Ontology slim and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the annotation of the estimated transcripts with Blastx, we found that the expression of some genes involved in carbohydrate metabolism were expressed two-fold more strongly in the virulent strain than in the precocious strain. Characteristically, genes related to proteins secreted from the apical complex, proteases, cell attachment proteins, mitochondrial proteins, and transporters were most strongly upregulated in the virulent strain. Interestingly, the expression of genes associated with cell survival, development, or proliferation was strongly upregulated in the precocious strain. These findings suggest that virulent strains survive long before invasion and invade actively/successfully into host cells, whereas proliferative processes appear to affect precocity.
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http://dx.doi.org/10.1016/j.meegid.2016.02.021DOI Listing
June 2016

Population, genetic, and antigenic diversity of the apicomplexan Eimeria tenella and their relevance to vaccine development.

Proc Natl Acad Sci U S A 2015 Sep 9;112(38):E5343-50. Epub 2015 Sep 9.

Pathology and Pathogen Biology, Royal Veterinary College, North Mymms, Hertfordshire, AL9 7TA, United Kingdom;

The phylum Apicomplexa includes serious pathogens of humans and animals. Understanding the distribution and population structure of these protozoan parasites is of fundamental importance to explain disease epidemiology and develop sustainable controls. Predicting the likely efficacy and longevity of subunit vaccines in field populations relies on knowledge of relevant preexisting antigenic diversity, population structure, the likelihood of coinfection by genetically distinct strains, and the efficiency of cross-fertilization. All four of these factors have been investigated for Plasmodium species parasites, revealing both clonal and panmictic population structures with exceptional polymorphism associated with immunoprotective antigens such as apical membrane antigen 1 (AMA1). For the coccidian Toxoplasma gondii only genomic diversity and population structure have been defined in depth so far; for the closely related Eimeria species, all four variables are currently unknown. Using Eimeria tenella, a major cause of the enteric disease coccidiosis, which exerts a profound effect on chicken productivity and welfare, we determined population structure, genotype distribution, and likelihood of cross-fertilization during coinfection and also investigated the extent of naturally occurring antigenic diversity for the E. tenella AMA1 homolog. Using genome-wide Sequenom SNP-based haplotyping, targeted sequencing, and single-cell genotyping, we show that in this coccidian the functionality of EtAMA1 appears to outweigh immune evasion. This result is in direct contrast to the situation in Plasmodium and most likely is underpinned by the biology of the direct and acute coccidian life cycle in the definitive host.
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http://dx.doi.org/10.1073/pnas.1506468112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4586875PMC
September 2015

DB-AT: a 2015 update to the Full-parasites database brings a multitude of new transcriptomic data for apicomplexan parasites.

Nucleic Acids Res 2015 Jan 20;43(Database issue):D631-6. Epub 2014 Nov 20.

Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, North 20, West 10 Kita-ku, Sapporo, Hokkaido 001-0020, Japan Global Station for Zoonosis Control, GI-CoRE, Hokkaido University, North 20, West 10 Kita-ku, Sapporo, Hokkaido 001-0020, Japan

The previous release of our Full-parasites database (http://fullmal.hgc.jp/) brought enhanced functionality, an expanded full-length cDNA content, and new RNA-Seq datasets from several important apicomplexan parasites. The 2015 update witnesses the major shift in the databases content with focus on diverse transcriptomes of the apicomplexan parasites. The content of the database was substantially enriched with transcriptome information for new apicomplexan parasites. The latest version covers a total of 17 species, with addition of our newly generated RNA-Seq data of a total of 909,150,388 tags. Moreover, we have generated and included two novel and unique datasets, which represent diverse nature of transcriptomes in individual parasites in vivo and in vitro. One is the data collected from 116 Indonesian patients infected with Plasmodium falciparum. The other is a series of transcriptome data collected from a total of 38 single cells of P. falciparum cultured in vitro. We believe that with the recent advances our database becomes an even better resource and a unique platform in the analysis of apicomplexan parasites and their interaction with their hosts. To adequately reflect the recent modifications and the current content we have changed the database name to DB-AT--DataBase of Apicomplexa Transcriptomes.
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http://dx.doi.org/10.1093/nar/gku1240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383994PMC
January 2015

Genomic analysis of the causative agents of coccidiosis in domestic chickens.

Genome Res 2014 Oct 11;24(10):1676-85. Epub 2014 Jul 11.

Wellcome Trust Sanger Institute, Genome Campus, Hinxton, Cambridgeshire CB10 1SA, United Kingdom;

Global production of chickens has trebled in the past two decades and they are now the most important source of dietary animal protein worldwide. Chickens are subject to many infectious diseases that reduce their performance and productivity. Coccidiosis, caused by apicomplexan protozoa of the genus Eimeria, is one of the most important poultry diseases. Understanding the biology of Eimeria parasites underpins development of new drugs and vaccines needed to improve global food security. We have produced annotated genome sequences of all seven species of Eimeria that infect domestic chickens, which reveal the full extent of previously described repeat-rich and repeat-poor regions and show that these parasites possess the most repeat-rich proteomes ever described. Furthermore, while no other apicomplexan has been found to possess retrotransposons, Eimeria is home to a family of chromoviruses. Analysis of Eimeria genes involved in basic biology and host-parasite interaction highlights adaptations to a relatively simple developmental life cycle and a complex array of co-expressed surface proteins involved in host cell binding.
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http://dx.doi.org/10.1101/gr.168955.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4199364PMC
October 2014

Characterization of Eimeria brunetti Isolated from a Poultry Farm in Japan.

J Vet Med Sci 2014 Jan 21;76(1):25-9. Epub 2013 Aug 21.

Nippon Institute for Biological Science, 9-2221-1 Shin-machi, Ome, Tokyo 198-0024, Japan.

None of anticoccidial vaccines (Trivalent TAM™, monovalent Neca™ and imported pentavalent Paracox(®)-5) contain Eimeria brunetti in Japan, which has not been regarded as a cause of coccidiosis, because of its low prevalence. However, we have recently reported the evidence of a high nationwide prevalence of this species. In this report, we describe the characteristics of E. brunetti which have never been clearly defined in Japan. Mortality rates and other disease characteristics caused by our strain (Nb strain) were similar to those reported previously in other studies. Despite great reduction of body weight gains among groups infected with over 1 × 10(3) oocysts, the intestinal lesions in the infected chickens were rather mild compared to previous studies. Sulfa drugs and lasalocid were so effective that the E. brunetti infection was almost completely blocked. Consequently, it is suggested that the characteristics of E. brunetti are various among the strains, but the pathogenicity of the Japanese Nb strain is enough strong to cause clinical coccidiosis.
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http://dx.doi.org/10.1292/jvms.13-0239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979942PMC
January 2014

Genetic analysis and development of species-specific PCR assays based on ITS-1 region of rRNA in bovine Eimeria parasites.

Vet Parasitol 2010 Nov 11;174(1-2):49-57. Epub 2010 Aug 11.

Nippon Institute for Biological Science, Shin-machi 9-2221-1, Ome, Tokyo 198-0024, Japan.

At present, morphological characteristics of oocyst is the only achievable method for the identification of bovine coccidia to the species level. In this study, the internal transcribed spacer 1 (ITS-1) region of ribosomal RNA genes of six bovine Eimeria species; E. alabamensis, E. auburnensis, E. bovis, E. cylindrica, E. ellipsoidalis and E. zuernii, were sequenced and analyzed the phylogenetic relationship among them. In pair-wise alignment, the sequences among the same species had high homology of over 90%. E. bovis and E. zuernii were closely related within the same cluster. This cluster and E. alabamensis were distant from major cluster of bovine coccidia that included E. auburnensis, E. cylindrica and E. ellipsoidalis. Species-specific PCR assays based on the amplification of the ITS-1 region were also developed to identify the 6 pathogens. The ITS-1 region of each Eimeria species had sufficient inter-specific sequence variation enough to design the primer sets that differentially amplified each target species. This PCR assay for the detection and differentiation of Eimeria parasite showed higher sensitivity when compared to the conventional oocyst-morphological examination. This is the first attempt for the identification of 6 bovine Eimeria parasites in the genomic level and may provide as useful methods for diagnosis and epidemiology of bovine coccidial infection.
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http://dx.doi.org/10.1016/j.vetpar.2010.08.001DOI Listing
November 2010

Detection of five avian Eimeria species by species-specific real-time polymerase chain reaction assay.

Avian Dis 2008 Dec;52(4):652-6

Nippon Institute for Biological Science, Shinmachi 9-2221-1, Ome, Tokyo 198-0024, Japan.

To detect five different avian Eimeria species, we applied the SYBR Green-based real-time polymerase chain reaction (PCR) assay for the diagnosis of field-isolated parasites by using their individual species-specific primer sets. The primer sets were originally designed for Eimeria acervulina, E brunetti, E necatrix, and E tenella based on the sequence of the internal transcribed spacer 1 region of ribosomal DNA, whereas for E. maxima the primer sets were derived from sequences reported previously. The detection limit of these assays was defined at 10(2) or 10(1) oocysts depending on species. Melting curves from the real-time PCR assay showed that each species has a single peak and specific melting temperature value. Fecal samples from 32 poultry farms, which were endemic for coccidiosis, were examined using this assay. The data showed that E. brunetti was found in 21 farms, E maxima and E. necatrix in 16 farms, E. tenella in 12 farms, and E. acervulina in eight farms. This survey revealed that E brunetti was highly prevalent in Japan. This technique is not only easy and rapid but also available to detect Eimeria species specifically; therefore, it can be a valuable tool for diagnostic work for chicken coccidiosis.
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http://dx.doi.org/10.1637/8351-050908-Reg.1DOI Listing
December 2008

Field basis evaluation of Eimeria necatrix-specific enzyme-linked immunosorbent assay (ELISA) for its utility in detecting antibodies elicited by vaccination in chickens.

J Vet Med Sci 2005 Sep;67(9):947-9

Nippon Institute for Biological Science, Tokyo, Japan.

Eimeria necatrix-specific ELISA, using a recombinant antigen (the cDNA-clone NP19 expressing protein), was utilized to detect antibodies against E. necatrix in breeder pullet flocks that had previously received an attenuated live vaccine to E. necatrix. Vaccinated flocks were discriminated significantly from non-vaccinated flocks by their antibody titers and antibody positive rates at 30-55 days post-vaccination. In addition, E. necatrix-oocysts were confirmed in fecal samples of vaccinated flocks using PCR in the case where the antibody positive rates rose. These findings implied that the vaccination prompted repeated infections, and consequently the chickens generated antibodies and secured their protection against virulent field-E. necatrix. Therefore, the ELISA was suggested to be a useful tool to estimate the immune state of chickens as a result of vaccination with a live E. necatrix-vaccine.
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http://dx.doi.org/10.1292/jvms.67.947DOI Listing
September 2005

Differential effects of interferon-gamma on production of trypanosome-derived lymphocyte-triggering factor by Trypanosoma brucei gambiense and Trypanosoma brucei brucei.

J Parasitol 2004 Aug;90(4):740-5

Division of Veterinary Science, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, 1-1, Gakuencho, Sakai, Osaka 599-8531, Japan.

Trypanosome-derived lymphocyte-triggering factor (TLTF) produced by Trypanosoma brucei brucei stimulates production of interferon-gamma (IFN-gamma) by CD8+ T cells, and it is reported that, in turn, IFN-gamma stimulates proliferation of T. b. brucei. We studied the role of TLTF in trypanosome proliferation using the Wellcome strain (WS) of Trypanosoma brucei gambiense and the ILtat 1.4 strain (IL) of T. b. brucei. Increase in the number of WS in infected rats is more rapid than IL and corresponds with comparatively higher levels of IFN-gamma. Production of IFN-gamma, as measured by protein and messenger RNA (mRNA) levels, was maintained by splenocytes from WS-infected rats, whereas levels decreased in IL-infected rats, accompanied by prolongation of infection. Expression of TLTF mRNA by in vitro-cultured WS was promoted in a dose-dependent fashion by addition of recombinant rat IFN-gamma at all concentrations tested. The addition of lower concentrations of IFN-gamma to cultured IL increased expression of TLTF mRNA, whereas, in contrast to WS, addition of 100 and 1,000 U/ml IFN-gamma decreased expression of TLTF by IL. These results show that unlike WS, elevated IFN-gamma concentrations lead to decreased TLTF production by IL. It is believed that decreased TLTF production in IL-infected rats leads to lowered IFN-gamma production, thereby slowing IL proliferation.
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http://dx.doi.org/10.1645/GE-211R1DOI Listing
August 2004
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