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Background/Aim: 1,5-Anhydro-d-fructose (1,5-AF, saccharide) and 1,5-anhydro-d-glucitol (1,5-AG) converted from 1,5-AF via the glycemic pathway have health benefits. However, this metabolism has not been sufficiently elucidated. To clarify the in vivo metabolism of 1,5-AF to 1,5-AG, porcine (blood kinetics) and human (urinary excretion) studies were conducted. Materials and Methods: Microminipigs were administrated 1,5-AF orally or intravenously. Blood samples were obtained to analyse the kinetics of 1,5-AF and 1,5-AG. Urine samples were collected from human subjects who had orally ingested 1,5-AF, and the amounts of 1,5-AF and 1,5-AG excreted in the urine were analysed. Results: In blood kinetics analysis, the time to the maximum concentration of 1,5-AF after intravenous administration was 0.5 h, whereas 1,5-AF was not observed after oral administration. The times to the maximum concentration of 1,5-AG after intravenous and oral administration were 1.5 h and 2 h, respectively. In urinary excretion, the concentration of 1,5-AG in urine rapidly increased after the administration of 1,5-AF, peaked at 2 h, whereas 1,5-AF was not detected. Conclusion: 1,5-AF was rapidly metabolized to 1.5-AG in vivo in swine and human.

Introduction Potential biomarkers for chronic seasonal heat stress in Kagoshima Berkshire pigs reared in the subtropical region were investigated by comparing the biomarker changes in the summer (a period of chronic heat stress) and winter (a thermoneutral period) seasons. Material and Methods Pigs were allocated to summer- and winter-finishing cohorts, 12 each. The evaluations included assessment of carcass traits and internal organs’ normality carried out at the time of slaughter, and measurement of biomarkers in whole blood: derivatives of reactive oxygen metabolites (d-ROMs) and biological antioxidant potential as markers of oxidative stress, and serum amyloid A and albumin/globulin (A/G) ratio as markers of acute and chronic inflammation, respectively. Results The summer-finished pigs reared under subtropical field conditions showed lower carcass quality than the winter-finished pigs, indicating a potential adverse effect of summer temperatures on the swine industry. Marginal changes were observed in d-ROMs and the A/G ratio between the summer- and winter-finishing cohorts. Conclusion The results demonstrate that d-ROMs and the A/G ratio could be used as sensitive markers for heat stress under field conditions.

Background/aim The number of vertebrae in swine varies from 19 to 23 and is associated with body size. Nuclear receptor subfamily 6 group A member 1 (NR6A1) is considered a strong candidate for affecting the number of vertebrae in swine. Wild boars, which uniformly have 19 vertebrae, have the wild type allele while multi-vertebrae European commercial pigs have the mutated allele. Our aim was to confirm the factor of the miniaturization. Materials and methods We examined vertebrae number and NR6A1 polymorphism in the Microminipig and three domestic breeds that vary in body size. Results The Microminipig had 19 or less vertebrae and a wild type NR6A1 genotype. Three domestic breeds had more than 21 vertebrae while the largest vertebrae number was observed in multi-vertebrae-fixed Large White. Heterozygous genotypes were observed in the middle-sized indigenous pig while homozygous NR6A1 mutations were observed in European commercial breeds. Conclusion NR6A1 could be a useful index for both miniaturizing and increasing pig body size.

There have been no reports of the prevalence of Eimeria spp. in poultry breeding farms in Japan unlike those of broiler farms. From 2017 to 2018, we examined the prevalence of Eimeria spp. on breeding farms in Japan by oocyst morphology and PCR analyses. A total of 143 samples was collected from 37 breeding farms in 21 prefectures of Japan. We detected oocysts of seven species at 34 of 37 breeding farms by PCR, and we identified E. brunetti at 51.5% of farms found to be positive for Eimeria. The differences in the identification of Eimeria spp. between the morphology and PCR assay methods of oocysts were pronounced for E. maxima and E. necatrix. We confirmed that molecular tools were more suitable for accurately estimating prevalence of Eimeria spp., and these findings suggest that E. brunetti could be widespread in Japan.

There have been no reports of the prevalence of Eimeria spp. in poultry breeding farms in Japan unlike those of broiler farms. From 2017 to 2018, we examined the prevalence of Eimeria spp. on breeding farms in Japan by oocyst morphology and PCR analyses. A total of 143 samples was collected from 37 breeding farms in 21 prefectures of Japan. We detected oocysts of seven species at 34 of 37 breeding farms by PCR, and we identified E. brunetti at 51.5% of farms found to be positive for Eimeria. The differences in the identification of Eimeria spp. between the morphology and PCR assay methods of oocysts were pronounced for E. maxima and E. necatrix. We confirmed that molecular tools were more suitable for accurately estimating prevalence of Eimeria spp., and these findings suggest that E. brunetti could be widespread in Japan.

The virulence of a type III Toxoplasma gondii strain isolated in Japan and designated here as TgCatJpGi1/TaJ was examined in mice and micro minipigs in this study. Despite its type III genotype, oral or intraperitoneal inoculation of cysts from it resulted in severe virulence in C57BL/6J and BALB/c mice. In contrast, mice inoculated with a high dose of TgCatJpGi1/TaJ tachyzoites showed no obvious clinical signs of infection, and all of them survived for >21 days post-inoculation. Furthermore, no clinical signs of infection were seen when micro minipigs were inoculated with 900 cysts. Interestingly, our allelic type screening of the virulence-related rop5, rop16, rop17, and rop18 genes, as based on restriction fragment length polymorphism analysis (RFLP), revealed that the RFLP patterns for TgCatJpGi1/TaJ were identical to those from nonvirulent type III parasites. These results suggest that TgCatJpGi1/TaJ possesses an unknown virulence factor or factors.

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction.

Thus far, Entamoeba species have been classified based on morphology such as the number of nuclei in mature cysts and their hosts. Using recently developed molecular tools, ruminant Entamoeba spp. are currently classified into four species/genotypes: E. bovis and Entamoeba ribosomal lineages (RL) 1, 2, and 4. However, the distribution or pathogenicity of ruminant Entamoeba has not been well documented. In the present study, we examined a total of 25 fecal and seven environmental samples collected from six farms in Japan from 2016 to 2017 by the floatation method and PCR and sequencing analyses. Consequently, we detected Entamoeba cysts in 18 of 25 cattle samples and four of the seven environmental samples, including soil and drinking water, by microscopic examinations. In sequential examinations, Entamoeba-positive cattle were found to shed cysts without any clinical symptoms for more than 8 months. By PCR for molecular identification, isolates in ten cattle and one soil sample were successfully sequenced and formed a cluster of E. bovis, which was separated from those of other Entamoeba species/genotypes such as RL1-4 in phylogenetic analysis. To our knowledge, this is the first report about E. bovis in Japan, and our results may implicate that E. bovis is not pathogenic.

To reveal the distribution of tick-borne parasites, we established a novel nested polymerase chain reaction (PCR) system to detect the most common agents of tick-borne parasitic diseases, namely Babesia, Theileria, and Hepatozoon parasites. We collected host-seeking or animal-feeding ticks in Kagoshima Prefecture, the southernmost region of Kyusyu Island in southwestern Japan. Twenty of the total of 776 tick samples displayed a specific band of the appropriate size (approximately 1.4-1.6kbp) for the 18S rRNA genes in the novel nested PCR (20/776: 2.58%). These PCR products have individual sequences of Babesia spp. (from 8 ticks), Theileria spp. (from 9 ticks: one tick sample including at least two Theileria spp. sequences), and Hepatozoon spp. (from 3 ticks). Phylogenetic analyses revealed that these sequences were close to those of undescribed Babesia spp. detected in feral raccoons in Japan (5 sequences; 3 sequences being identical), Babesia gibsoni-like parasites detected in pigs in China (3 sequences; all sequences being identical), Theileria spp. detected in sika deer in Japan and China (10 sequences; 2 sequences being identical), Hepatozoon canis (one sequence), and Hepatozoon spp. detected in Japanese martens in Japan (two sequences). In summary, we showed that various tick-borne parasites exist in Kagoshima, the southern region in Japan by using the novel nested PCR system. These including undescribed species such as Babesia gibsoni-like parasites previously detected in pigs in China. Importantly, our results revealed new combinations of ticks and protozoan parasites in southern Japan. The results of this study will aid in the recognition of potential parasitic animal diseases caused by tick-borne parasites.

In the production and management of beef and dairy cattle, controlling diarrhea is one of the important concerns. Pathogenic agents of the disease, protozoan parasites including Cryptosporidium spp., are difficult to control, making prevention, diagnoses, and treatment of diarrhea. In the present study, we investigated a farm with a history of calf deaths over a period of 10 years in order to determine the cause of disease and to clarify the detailed distribution of the pathogens. In four examined calves that were reared in calf pens, all were positive with Cryptosporidium and/or Giardia, while the other breeding stock and adult cattle were negative. Molecular analyses revealed that the isolates from calves were C. parvum subtype IIaA15G2R1 as a zoonotic and G. intestinalis assemblage E. Other pathogenic bacteria and diarrhea-causing viruses were not detected. After treating the calf pens with boiling water and milk of lime (Ca[OH]2), oocysts of C. parvum and cysts of G. intestinalis were not found and no additional calves died. This is the first report to describe the mixed infection of both parasites in Japan.

A tumor necrosis factor receptor-associated factor-type zinc finger domain containing protein 1 (TRAFD1) is a negative feedback regulator that controls excessive immune responses in vertebrates. The sequence of tick hemolymph TRAFD1 from the hard tick Haemaphysalis longicornis (HlTRAFD1) was analyzed after identification and cloning from the expressed sequence tag database. RT-PCR and Western blot analyses showed that HlTRAFD1 transcript and protein levels after blood feeding were present in all developmental stages, and the transcript level was consistently high in all organs examined from adult female ticks upon engorgement. Knockdown of HlTRAFD1 gene by RNA interference did not affect blood feeding or oviposition. However, HlTRAFD1 silencing affected the expression of the longicin gene, a defensin-like molecule, but not the lysozyme gene. Moreover, the survival rate of HlTRAFD1-silenced ticks was lower, and the number of E. coli was higher in the hemolymph and plasmatocytes after E. coli injection compared to the control group. These results suggested that HlTRAFD1 strongly affected both the humoral and cellular immunity of ticks.

Toxoplasma gondii is classified into 16 haplogroups based on a worldwide genotyping study of the parasite. However, only a few isolates from Japan were included in this analysis. To conduct more precise genotyping of T. gondii, we examined the genotypes of Japanese isolates in this study. DNA sequences of 6 loci were determined in 17 Japanese isolates and compared with those of strains of 16 haplogroups. As a result, Japanese isolates were classified into four groups. We investigated the virulence of some Japanese isolates and found a highly virulent strain in mice, comparable to that of RH strain, although this Japanese isolate was sister to strains of haplogroup 2, which show moderate virulence in mice. We further investigated whether this high virulence isolate had different virulence mechanism and strategy to adapt to Japanese host from other strains by comparing the virulence-related genes, ROP5, 18 and the immunomodulatory gene, ROP16 of the isolate with those of archetypical strains (GT1, ME49 and VEG). This analysis indicated the high virulence of the isolate in mice was partly explained by gene sequences of ROP5 and ROP16. These findings lead to the elucidation of biodiversity of T. gondii and have potential to optimize the diagnostic protocol.

The Toxoplasma gondii strain TgCatJpTy1/k-3 (K-3), isolated from a stray cat in Tokyo, Japan, is categorized as a type II genotype. Since the K-3 strain is empirically known to form relatively larger cysts and exhibit weak pathogenesis in a mouse, it could serve as a useful model organism to study chronic T. gondii infection in the host. However, a detailed biological characterization of this strain had not been performed. In this study, we thoroughly assessed the K-3 strain in vivo using a mouse model. Tests indicated that pathogenicity of the K-3 strain was lower than that of the PLK strain, a clonal laboratory strain with a moderately pathogenic type II genotype. Further, cyst sizes of the K-3 strain were significantly larger than those of the PLK strain. Interestingly, K-3 cyst sizes in T. gondii-resistant ICR mice were larger than those in T. gondii-susceptible C57BL/6N mice. Our study suggests that the K-3 strain is suitable to study T. gondii cystogenesis and chronic infection, which are currently difficult to analyze using cell-adopted T. gondii strains.

Murine Eimeria spp. have been used as effective experimental models of disease instead of large mammalian hosts such as cattle. We here examine drug susceptibility of the uncharacterized murine intestinal protozoan parasite, Eimeria krijgsmanni. The effectiveness of different treatments against infection of E. krijgsmanni was examined for suppression of oocyst shedding: ST mixture ST mixture, pyrimethamine, Ektecin and toltrazuril. ST mixture and pyrimethamine did not suppress oocyst shedding effectively. Although therapeutic efficacy of Ektecin was demonstrated, the dose required was larger than that for cattle and chickens. Oocyst shedding was only completely suppressed completely by continuous administration of toltrazuril. Furthermore, it was confirmed through morphological examination that early developmental stage zoites appeared in host epithelial cells during and following treatment by toltrazuril, and toltrazuril could not eliminate residual zoites in epithelial cells. E. krijgsmanni may be relatively resistant to these anti-coccidian agents and might therefore have different characteristics that differ from other coccidia with regard to drug susceptibility.

Haemaphysalis longicornis is an important vector of various pathogens in domestic animals and humans. The tick is a unique species with bisexual and parthenogenetic races. Although mating induces oocyte development, it is possible in the parthenogenetic race to complete oogenesis without copulation. Here we examined the developmental process of oocytes from unfed to the oviposition period in parthenogenetic H. longicornis. We classified the developmental stages of oocytes into five stages: stage I, germinal vesicle occupies more than half of the cytoplasm; stage II, germinal vesicle occupies less than half of the cytoplasm; stage III, germinal vesicle migrates from the center in the oocyte to the vicinity of the pedicel cells; stage IV, the cytoplasm is filled with yolk granules of various sizes; stage V, the cytoplasm is occupied by large yolk granules. Oocytes at the unfed period were undeveloped and classified as stage I. Stage I and II oocytes were observed at the rapid feeding period, indicating that oocyte development began after the initiation of blood feeding. All developmental stages of oocytes were observed at the pre-oviposition period. At 10 days after the beginning of the oviposition period, the ratios of stage I and II oocytes were higher than those of the previous period, suggesting that the ovarian development and activity may be continuing. Based on these findings, we propose classification criteria for the oocyte development in the parthenogenetic H. longicornis. The criteria will be useful for understanding the mechanisms of tick reproduction and transovarial transmission of pathogens.

Western blotting of adult female ticks during blood-feeding. Protein lysates were extracted from adult female ticks during the different stages of blood-feeding. Protein lysates were run on 12% SDS-PAGE gel before being transferred to PVDF membranes and subjected to Western blotting. Mouse tubulin antiserum was used as control. Leftmost lane indicates markers for molecular weight. (PDF 253 kb)

The modeled tertiary structures of HlGST (a) and HlGST2 (b). The model is based on template c1b8xA [48] constructed using PHYRE2 software [49]. Green indicates the N-terminal domain containing the GSH binding site (orange), while red indicates the C-terminal domain containing the substrate binding site (blue). The mu-loop is indicated by bluish green. (PDF 193 kb)

P- and t-values of GST transcription profiles. (DOCX 40 kb)

RT-PCR (a) and Western blotting (b) of knockdown ticks. Ticks were silenced by injecting 1 Îźg of double-stranded RNA (dsRNA) per tick. a Total RNA was extracted from whole 4-day fed GST and EGFP knockdown ticks. cDNA was synthesized and subjected to RT-PCR. PCR products were run on 1.5% TAE agarose gel and stained with ethidium bromide. Actin was used as loading control. b Protein lysates were extracted from whole 4-day-fed HlGST, HlGST2, HlGST1/2, and EGFP knockdown ticks. Protein lysates were run on 12% SDS-PAGE gel before being transferred to polyvinylidene difluoride (PVDF) membranes and subjected to Western blotting. Mouse tubulin antiserum was used as control. (PDF 200 kb)

Nucleotide and deduced amino acid sequences of HlGST (a) and HlGST2 (b) of Haemaphysalis longicornis. Start and stop codons are underlined. Predicted glutathione and substrate binding sites are shaded in black and gray, respectively. The putative polyadenylation signal, AATAAA, is double underlined. (PDF 53 kb)

Effect of HlGST and/or HlGST2 knockdown on ticks. (DOCX 36 kb)

The Iriomote cat (IC), Prionailurus bengalensis iriomotensis, and the Tsushima leopard cat (TLC), Prionailurus bengalensis euptilurus, are endangered subspecies of leopard cats in Japan. In addition to habitat destruction and road kills, infectious diseases may threaten their populations, and infection with arthropod-borne pathogens has been reported in both subspecies. Infestations with ectoparasites, especially ticks, have frequently been observed in ICs and TLCs. In the present study, ticks collected from captured ICs and TLCs between November 2011 and January 2012 were morphologically identified and the prevalence of the pathogens Bartonella sp., Babesia sp., Ehrlichia sp., Anaplasma sp., Hepatozoon sp., and hemoplasmas in the ticks was molecularly evaluated. The ticks Haemaphysalis longicornis, H. hystricis, and Amblyomma testudinarium were obtained from ICs, and H. megaspinosa, Ixodes tanuki, H. campanulata, and A. testudinarium were collected from TLCs. The pathogens Hepatozoon felis, Babesia sp., and Anaplasma bovis were detected in ticks obtained from ICs, while H. felis. Babesia sp., Ehrlichia sp., E. muris, ‘Candidatus Mycoplasma haemominutum’, and Bartonella henselae were found in ticks from TLCs. To protect and conserve these endangered animals, continuous monitoring and additional surveys will be necessary to understand the role of ticks as disease vectors in Japanese wildcats.

Background Ticks are obligate hematophagous parasites important economically and to health. Ticks consume large amounts of blood for their survival and reproduction; however, large amounts of iron in blood could lead to oxidative stress. Ticks use several molecules such as glutathione S-transferases (GSTs), ferritins, and peroxiredoxins to cope with oxidative stress. This study aimed to identify and characterize the GSTs of the hard tick Haemaphysalis longicornis in order to determine if they have a role in coping with oxidative stress. Methods Genes encoding GSTs of H. longicornis were isolated from the midgut CDNA library. Genes have been cloned and recombinant GSTs have been expressed. The enzymatic activities, enzyme kinetic constants, and optimal pH of the recombinant GSTs toward 1-chloro-2,4-dinitrobenzene (CDNB) were determined. The gene transcription and protein expression profiles were determined in the whole ticks and internal organs, and developmental stages using real time RT-PCR and Western blotting during blood feeding. The localization of GST proteins in organs was also observed using immunofluorescent antibody test (IFAT). Results We have isolated two genes encoding GSTs (HlGST and HlGST2). The enzymatic activity toward CDNB is 9.75 ± 3.04 units/mg protein for recombinant HlGST and 11.63 ± 4.08 units/mg protein for recombinant HlGST2. Kinetic analysis of recombinant HlGST showed Km values of 0.82 ± 0.14 mM and 0.64 ± 0.32 mM for the function of CDNB and GSH, respectively. Meanwhile, recombinant HlGST2 has Km values of 0.61 ± 0.20 mM and 0.53 ± 0.02 mM for the function of CDNB and GSH, respectively. The optimum pH of recombinant HlGST and recombinant HlGST2 activity was 7.5–8.0. Transcription of both GSTs increases in different developmental stages and organs during blood-feeding. GST proteins are upregulated during blood-feeding but decreased upon engorgement in whole ticks and in some organs, such as the midgut and hemocytes. Interestingly, salivary glands, ovaries, and fat bodies showed decreasing protein expression during blood-feeding to engorgement. Varying localization of GSTs in the midgut, salivary glands, fat bodies, ovaries, and hemocytes was observed depending on the feeding state, especially in the midgut and salivary glands. Conclusions In summary, a novel GST of H. longicornis has been identified. Characterization of the GSTs showed that GSTs have positive correlation with the degree and localization of oxidative stress during blood-feeding. This could indicate their protective role during oxidative stress. Electronic supplementary material The online version of this article (10.1186/s13071-018-2667-1) contains supplementary material, which is available to authorized users.

Transferrin is known to be an iron transporter in vertebrates and several arthropods. Iron from host blood is essential for ovarian development in blood-sucking arthropods. However, tick transferrin has been identified in only a few species, and its function has yet to be elucidated, resulting in incomplete understanding of iron metabolism in ticks. Here, we investigated the transfer of host-derived transferrin in the hard tick Haemaphysalis longicornis using immunological methods. Western blot showed that host-derived transferrin was maintained in all developmental stages of ticks up to 28 days after engorgement and was detected in the midgut and the ovary of adult females following blood feeding. However, no host-derived transferrin was detected in eggs after laying or in larvae after hatching, indicating that host-derived transferrin is not transferred to offspring transovarially. Indirect immunofluorescent antibody testing showed the localization of host-derived transferrin in digestive cells of the midgut and oocytes of the ovary from engorged adult females. These results suggest that host-derived transferrin is transferred to the ovary through the midgut and the hemolymph, and raise the possibility of the function of host-derived transferrin as an iron source in the ovary, providing additional insight on iron metabolism in ticks.

The Haemaphysalis longicornis longicin P4 peptide is an active part peptide produced by longicin which displays bactericidal activity against both Gram-negative and Gram-positive bacteria and other microorganisms. In the present study, the effect of the longicin P4 peptide on the infectivity of Toxoplasma gondii parasites was examined in vitro. Tachyzoites of T. gondii incubated with longicin P4 had induced aggregation and lost the trypan blue dye exclusion activity and the invasion ability into the mouse embryonal cell line (NIH/3T3). Longicin P4 bound to T. gondii tachyzoites, as demonstrated by fluoresce microscopic analysis. An electron microscopic analysis and a fluorescence propidium iodide exclusion assay of tachyzoites exposed to longicin P4 revealed pore formation in the cellular membrane, membrane disorganization, and hollowing as well as cytoplasmic vacuolization. The number of tachyzoites proliferated in mouse macrophage cell line (J774A.1) was significantly decreased by incubation with longicin P4. These findings suggested that longicin P4 conceivably impaired parasite membranes, leading to the destruction of Toxoplasma parasites in J774A.1 cells. Thus, longicin P4 is an interesting candidate for antitoxoplasmosis drug design that causes severe toxicity to T. gondii and plays an important role in reducing cellular infection. This is the first report showing that longicin P4 causes aggregation and membrane injury of parasites, leading to Toxoplasma tachyzoite destruction.

Toxoplasma gondii is an obligate intracellular protozoan parasite that invades a wide range of host cells. The parasite releases a large variety of proteins from a secretory organelle, microneme, and the secretion is essential for the parasite invasion. We cloned a secreted protein with an altered thrombospondin repeat of Toxoplasma gondii (TgSPATR), which was the homologue of Plasmodium SPATRs. Immunofluorescence double staining experiment revealed that TgSPATR was co-localized with a microneme protein, MIC2, and immuno-electron microscopic (IEM) analysis detected TgSPATR in the microneme-like structure. TgSPATR secretion was induced by ethanol, while an intracellular Ca(2+) chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM), suppressed the ethanol-induced secretion, suggesting the secretion was Ca(2+)-dependent, similarly to known microneme proteins. Furthermore, TgSPATR, existed on outer surface of the parasites, was detected by incomplete membrane permeabilization by saponin and immunofluorescent antibody test (IFAT). Both TgSPATR and MIC2 were detected on outer surface of extracellular parasites, but not of intracellular single parasites, suggesting they were similarly secreted during early stages of parasite invasion. Therefore, TgSPATR is probably new member of microneme protein and maybe involved in parasite invasion.

Although parasite-infected host cells become resistant to apoptosis, uninfected bystander cells undergo apoptosis during Toxoplasma gondii infection. The Programmed Cell Death 5 ( TgPDCD5 ) gene, a homologue of the human apoptosis-related molecule, was cloned from a T. gondii full-length cDNA database and subsequently characterized. The native TgPDCD5 was located in the cytosol and also detected in the secreted fraction. Immuno-electron microscopic analysis showed TgPDCD5 was primarily located close to the rhoptries or vesicle-like structures near the surface membrane of the parasite. Studies using recombinant TgPDCD5 (rTgPDCD5) demonstrated that host cells internalize the molecule in a heparan sulfate proteoglycan-binding motif-dependent manner. Furthermore, the addition of rTgPDCD5 to culture medium resulted in the enhancement of host-cell apoptosis triggered by etoposide in macrophage cell line J774A.1 and leukemic cell line HL-60 cells. Additionally, rTgPDCD5 induced apoptosis in J774A.1 cells in the presence of IFN-γ. This report is the first to identify a parasitic molecule of T. gondii that has a pro-apoptotic effect on host cells.

Recently, we have demonstrated the utility of Eimeria krijgsmanni as a novel mouse eimerian parasite for elucidating the biological diversity. The parasite showed notable infectivity to mice with various levels of immune status and susceptibility to antimicrobial agents including coccidiostat. However, the detailed lifecycle of E. krijgsmanni had not yet been determined and this information was lacking in discussion of previous findings. In the present study, we clarified the morphological characteristics of E. krijgsmanni and its lifecycle in normal mice, and examined the effects in immunodeficient mice and lifecycle stage for challenge infections after the primary inoculation. In immunocompetent mice, the lifecycle consisted of four asexual stages and the sexual sages followed by formation of oocysts during the prepatent periods. Interestingly, the second-generation meronts were detected in all observation periods after the disappearance of the other stages. For the challenge infection of immunodeficient mice, all developmental stages except for the second generation meronts were temporarily vanished. This finding suggests a "rest" or marked delay in development and a "restart" of the promotion toward the next generations. The second generation meronts may play an important role in the lifecycle of E. krijgsmanni.

Theirelia orientalis is a tick-borne haemoprotozoan parasite, and infection with this parasite is one of the most important diseases for grazing cattle. Co-infection of cattle with different genotypes of T. orientalis often occurs. In this study, we investigated the temporal dynamics of genotypes in cattle in a year-round grazing system in Japan. Genotype-specific PCR assays to determine major piroplasm surface protein (MPSP) genotypes (types 1 to 5) of T. orientalis were performed by using time-course blood samples collected from grazing cattle and ticks in a pasture. All 20 cattle investigated in this study were infected with T. orientalis. By using genotype-specific PCR, we detected the combination of genotypes of T. orientalis (types 1 to 5) from each cattle. These multiple genotypes of T. orientalis were also confirmed in ticks. Notably, each genotype of T. orientalis in cattle was temporally detected from cattle and more variable genotypes were found in summer. The observed temporal dynamics of the MPSP genotypes of T. orientalis in cattle could be explained by host immunity against the parasites or genetic recombination of parasite in ticks.

Ornithodoros moubataticks were fed on blood infected withBabesia equi. However, the parasites were quickly cleared as evidenced by the disappearance ofB. equi-specific ribosomal RNA from the ticks. We hypothesized that if theBabesiaparasite can escape midgut-associated barriers a non-vector tick can become infected withBabesia. To test this hypothesis,B. equiparasite-infected blood fromin vitroculture was injected into the haemocoel of ticks.B. equi-specific rRNA was surprisingly detected 45 days after injection even in the eggs.Babesia-free dogs were infested withO. moubataticks that were infected by inoculation withB. gibsoni-infected red blood cells. Parasitaemia and antibody production against Bg-TRAP ofB. gibsoniincreased gradually. These results indicate thatO. moubatamay be a useful vector model forBabesiaparasites and also a very important tool for studies on tick immunity againstBabesiaparasites and tick-Babesiainteractions.

Ticks are well-known vectors of various pathogens but migration of the pathogens in the tick midgut is not fully understood. In the present study, the fate of microbes in the midgut of Ornithodoros moubata was observed using green fluorescent protein (GFP)-expressing Escherichia coli. Fluctuations in the percentage of hemocytes in the hemolymph (Hc) and expression of an antimicrobial peptide, defensin, in the midgut was also investigated. Most E. coli gradually disappeared in the midgut after ingestion fluctuations in Hc coincided with the changes. Expression of defensin was also confirmed and slightly up-regulated after E. coli ingestion. Moreover, it was demonstrated that E. coli can not pass through the tick midgut epithelium after ingestion by the hemolymph cultures. It is known that various pathogens and host immunoglobulins ingested with a blood meal can enter into the hemocoel, which suggests the presence of unique and complex passage mechanisms for each molecule and organism. The results obtained here help to clarify that digestion enzymes is an important function of the tick midgut to protect against invading molecules and organisms.

We evaluated the growth inhibitory effect of triclosan, which has recently been reported to inhibit the growth of Plasmodium species and Toxoplasma gondii, on bovine and equine Babesia parasites in in vitro cultures The growth of Babesia bovis and B. bigemina was significantly inhibited in the presence of 100 microg/ml of triclosan, while B. caballi and B. equi were susceptible to as low as 50 microg/ml. Babesia bigemina and B. caballi were completely cleared as early as on the first and second day of the treatment, respectively. These parasites did not exhibit any growth in the subsequent five-day period of subculture without triclosan. Drug-treated parasites appeared pycnotic and atypically shaped, and ultrastructurally showed pronounced vacuolations, leading to complete destruction of parasites. Light microscopy showed that used concentrations of triclosan showed no toxicity against the host cells. The results suggest that triclosan can be used for chemotherapy of babesiosis.

Three antimalarial drugs, artesunate, pyrimethamine, and pamaquine, were evaluated for their growth-inhibitory effects against Babesia equi and Babesia caballi in in vitro culture. B. equi was more resistant to pyrimethamine than B. caballi. B. equi was also found to be more sensitive to artesunate and pamaquine than B. caballi . Of the three compounds, pyrimethamine gave the most promise for in vivo effectiveness.

Babesia microti, a hemoprotozoan parasite of rodents, is also important as a zoonotic agent of human babesiosis. The Maltese cross form, which consists of four masses in an erythrocyte, is characteristic of the developmental stage of B. microti . Monoclonal antibody (MAb) 2-1E, which specifically recognizes the Maltese cross form of B. microti , has been described previously. In the present study, we examined the roles of the Maltese cross form during the infectious course of B. microti in mice. The number of the Maltese cross form increased in the peripheral blood of infected mice prior to the peak of parasitemia. With confocal laser scanning microscopy, MAb 2-1E was found to be reactive with the ring form, with the parasites undergoing transformation to the Maltese cross form and subsequent division, and also with extracellular merozoites. Furthermore, the Maltese cross form-related antigen (MRA) gene was isolated from a B. microti cDNA library by immunoscreening with MAb 2-1E, and the nucleotide sequence was determined. Genomic analyses indicated that the MRA gene exists as a single-copy gene in B. microti . Immunization of mice with recombinant MRA induced significant protective immunity against B. microti infection. These findings indicate that the Maltese cross form plays important roles in both the development of parasitemia and the protective response against the infection.

Murine Eimeria spp. have been used as effective models of disease instead of large mammalian hosts such as cattle. We attempted to establish in vivo and in vitro assays using a murine intestinal protozoan, Eimeria krijgsmanni, which we previously isolated, to test anti-parasitic agents. Consequently, when mice were treated with sulfur drugs or toltrazuril, which are commercially available for livestock. Furthermore, sporulated oocysts and excysted sporozoites of E. krijgsmanni were treated with naturally occurring substances (lactoferrin, longicin, and curcumin). Although exposure to these substances did not affect oocyst infectivity, sporozoite viability decreased by 60% with longicin. However, direct injection of sporozoites treated with longicin into mice ceca did not result in any changes in the oocyst shedding pattern compared with control mice. The results suggest that E. krijgsmanni could be resistant to these anti-parasitic agents and might therefore have different characteristics to other apicomplexan parasites.

The adaptation and immunogenisity of Cryptosporidium parvum isolated from Siberian chipmunks (SC1 strain) in immunocompetent (ICR) mice were examined. The oocysts were received to the severe combined immunodeficiency (SCID) mice by repeated passage. The oocysts collected from the 18th SCID mice were inoculated to 5 ICR mice. The mice began to shed oocysts from 6 days after inoculation, the patency was 5 days, and the maximum oocysts per gram of feces (OPG) value was 104. The maximum of OPG value was gradually increased by successive passage, and finally that in the 22nd mice reached 106 (patency: 11 days). It is considered that these results indicate completion of their adaptation to ICR mice. To examine the immunogenicity of C. parvum to ICR mice, 8 groups of 5 mice each were inoculated with 1.3 × 106 oocysts of SC1 strain, which were collected after adaptation to SCID mice. All groups shed oocysts from 6th day, and their patency was from 8 to 12 days. On the 21st day after the primary infection, these mice were challenged with 1.3 × 106 oocysts. No oocysts shed from any groups, although 2 control groups shed oocysts from the 6th day, and their OPG values were more than 106. These results suggest that this strain has strong immunogenicity against ICR mice. Therefore, the immunological healthy mice were considered a useful experimental model to investigate immunological and drug treatments in the strain of C. parvum.

In Haemaphysalis longicornis, secretions of the male accessory genital glands were regenerated by re-feeding for 3 or 4 days, although the secretions were almost completely released during the first copulation. It was also shown that spermatogenesis continued during re-feeding, since prospermia (elongated spermatids) were deposited in the seminal vesicle. A potent male seeks a receptive female on the host for copulation. The movement of males to different attachment sites occurred between the third and fourth day of re-feeding, and completely re-fed males (for 4 days) were able to copulate successfully. Spermatogenic cells, ranging from spermatogonia at the anterior end to prospermia at the posterior end, were found in fed males. Degeneration of spermatocytes at the great growth phase and developing spermatids prior to final development of prospermia were seen in virgin males without re-feeding after the first meal. Fully elongated spermatids (prospermia) appeared morphologically normal up to 10 days after the first feeding. Degeneration of spermatocytes and developing spermatids occurred from the second day and was almost complete by the fourth day. The degenerating cells shrank, became electron-dense, and finally died. A reduction in secretions of the four lobes of the accessory glands occurred during the 10 days after feeding.

In a Prostriata, Ixodes ovatus Neumann (Acari: Ixodidae), the fate and ultrastructural changes of male germ cells were observed during their reproductive behavior including copulation and feeding. Before copulation, male germ cells in the male seminal vesicle were prospermia. After copulation, the timing of spermateleosis and ultrastructural changes during spermateleosis were the same as those in other ixodids. In I. ovatus, endospermatophores were fragmented in the cervical vagina, although they occur within the receptaculum seminis in Metastriata. After fragmentation of endospermatophores, the male germ cells reached a common oviduct, where they were stored for at least 30 days. On day 30 after copulation, the ultrastructure of the male germ cells in the common oviduct was the same as on day 2 after copulation. On the other hand, the male germ cells reached the oviducts on day 4 after engorgement (corresponding to day 9 after copulation). This indicates that feeding advances ascent of male germ cells.

The male accessory genital glands of Haemaphysalis longicornis are a complex of glands consisting of the single dorso-median and medio-ventral lobes and paired antero-ventral, latero-ventral, dorso-lateral, postero-lateral, and postero-ventral lobes. The epithelia of each lobe produce various secretory granules during feeding and these secretions have important functions in the formation of the spermatophore. Some of the accessory gland secretions form the spermatophore as a bag of male germ cells, and others are packed in the spermatophore and then transferred into the female genital tract. The fate of the accessory gland secretions was elucidated by comparison between the ultrastructure of the male accessory genital glands and the spermatophore, and the roles of these secretions are discussed. The characteristic fine structures of each lobe and 24 types of secretory granules were observed in the whole accessory glands. It was shown morphologically that two layers of the ectospermatophore were derived from the antero-ventral and postero-lateral (=dorso-lateral) lobes and three types of granular contents in the spermatophore from the postero-ventral, latero-ventral and medio-ventral lobes. These findings support the results of histochemical previous observations, and more accurate information on the derivation of substances in the formation of the spermatophore were obtained.

Copulation in ticks is completed by the insertion of the spermatophore into the female genital aperture by a male. The endospermatophore, a cord-like structure and contents are packed in the ectospermatophore of the completed spermatophore. The endospermatophore extrudes just after insertion of the tip of the spermatophore. Only the endospermatophore enters the female genital tract, and the ectospermatophore remains outside the female body. The extrusion is observed in vitro in Haemaphysalis longicornis at various concentrations of NaCl solution: the process is accelerated in less concentrated solutions. The cord-like structure and the endospermatophore finally receive contents extruded from the ectospermatophore. The tip of the cord-like structure connects to the surface of the endospermatophore, and together form a loop after extrusion. Ultrastructural observations confirmed that the ectospermatophore wall is composed of four layers, and the contents consist of male germ cells and three types of secretions from the male accessory genital glands. As in other ticks the male germ cells are elongated spermatids in spermatophores just after formation and extrusion. Adlerocysts described in other ticks are not found in the spermatophore of H. longicornis.

A rnale tick gives elongated spermatids to a female, that is, spermatogenesis within testes completes itself until elongated spermatids. And then elongated spermatids develop into mature sperms after they enter into the female genital tract via the spermatophore. In the present study, ultrastructural observations of spermatogenic cells were made at each stage of spermatogenesis in male Haemphysalis longicornis. Spermatogenic cells from spermatogonia to primary spermatocytes at the middle of their great growth phase were found in testes of unfed males. Subsequently, testes of H. longicomis are developed by feeding at their adult stage. The testes, or spermatogenic cells, were developed gradually until the 5-day feeding period was complete (the complete feeding stage). Spermatogenic cells at all stages from spermatogonia to elongated spermatids were contained in the testes of completely fed males. Spermatogenic cells were packed in cysts. Elongated spermatids which had been completed at the posterior ends of testes were made spermiation with the breakdown of the cyst cells. The spermatids were carried out of the testes through the central lumen. The cyst cells formed the lumen, and had some microvilli on their free surface.