Synthesis of the Carbohydrate Moiety of Glycoproteins from the Parasite Echinococcus granulosus and Their Antigenicity against Human Sera.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the carbohydrate moiety of glycoprotein from Echinococcus granulosus have been accomplished. Trisaccharide Galß1-3Galß1-3GalNAcα1-R (A), tetrasaccharide Galα1-4Galß1-3Galß1-3GalNAcα1-R (B), and pentasaccharide Galα1-4Galß1-3Galß1-3Galß1-3GalNAcα1-R (C), (R = biotinylated probe) were synthesized by stepwise condensation and/or block synthesis by the use of 5-(methoxycarbonyl)pentyl 2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. The synthesis of the tetrasaccharide and the pentasaccharide was improved from the viewpoint of reducing the number of synthetic steps and increasing the total yield by changing from stepwise condensation to block synthesis. Moreover, hexasaccharide E, which contains the oligosaccharide sequence which occurs in E. granulosus, was synthesized from trisaccharide D. We examined the antigenicity of these five oligosaccharides by an enzyme-linked immunosorbent assay (ELISA). Although compounds of C-E did not exhibit antigenicity against cystic echinococcosis (CE) patient sera, compounds B, D, and E showed good serodiagnostic potential for alveolar echinococcosis (AE).

A novel nairovirus associated with acute febrile illness in Hokkaido, Japan.

The increasing burden of tick-borne orthonairovirus infections, such as Crimean-Congo hemorrhagic fever, is becoming a global concern for public health. In the present study, we identify a novel orthonairovirus, designated Yezo virus (YEZV), from two patients showing acute febrile illness with thrombocytopenia and leukopenia after tick bite in Hokkaido, Japan, in 2019 and 2020, respectively. YEZV is phylogenetically grouped with Sulina virus detected in Ixodes ricinus ticks in Romania. YEZV infection has been confirmed in seven patients from 2014-2020, four of whom were co-infected with Borrelia spp. Antibodies to YEZV are found in wild deer and raccoons, and YEZV RNAs have been detected in ticks from Hokkaido. In this work, we demonstrate that YEZV is highly likely to be the causative pathogen of febrile illness, representing the first report of an endemic infection associated with an orthonairovirus potentially transmitted by ticks in Japan.

Synthesis of the Non Reducing End Oligosaccharides of Glycosphingolipids from Ascaris suum.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the non reducing end oligosaccharides of glycosphingolipids from Ascaris suum have been accomplished. Galα1→3GalNAcß1→OR (1), Galß1→3Galα1→3GalNAcß1→OR (2), Galß1→6Galα1→3GalNAcß1→OR (3), Galß1→6(Galß1→3)Galα1→3GalNAcß1→OR (4) and GlcNAcß1→6Galß1→6(Galß1→3)Galα1→3GalNAcß1→OR (5) (R = biotinylated probe) were synthesized by stepwise condensation (1-4) and block synthesis (5) using 5-(methoxycarbonylpentyl) 2-O-benzoyl-3-O-2-napthylmethyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-ß-D-galactopyranoside (12) as a common precursor. Compound 12 was converted into two kinds of glycosyl acceptors and was condensed with suitable galactosyl donors, respectively.

Case Report: Clinical Features of a Case of Suspected Borrelia miyamotoi Disease in Hokkaido, Japan.

We herein report a case of suspected Borrelia miyamotoi disease in Hokkaido, Japan. The patient complained of lassitude, arthralgia, and high fever after a tick bite. Furthermore, at the time of consultation, the patient exhibited momentary loss of consciousness and low blood pressure. Laboratory tests revealed elevation of liver enzymes, thrombocytopenia, and increased C-reactive protein. Seroconversion to B. miyamotoi glycerophosphoryl diester phosphodiesterase antigen suggested the patient was infected with a relapsing fever group Borrelia species.

Diagnosis of canine Echinococcus multilocularis infections by copro-DNA tests: comparison of DNA extraction techniques and evaluation of diagnostic deworming.

The use of copro-DNA detection methods for the diagnosis of canine Echinococcus multilocularis infection was evaluated with a focus on DNA extraction techniques: two commercial kits and a modified alkaline-sodium dodecyl sulfate (SDS) technique. Dog feces (0.2 g) mixed with a protoscolex or with 1 or 10 eggs of E. multilocularis were subjected to DNA detection following extraction by these methods. DNA was extracted from all protoscolex samples by all methods, but success for samples with eggs depended on extraction technique with the modified technique showing success on all samples. Following experimental infection of dogs, copro-DNA was successfully extracted from fecal samples (0.2 g) of dogs in the patent period by all methods. In the prepatent period, PCR testing of feces subsamples (0.2 g) extracted by each technique was positive at a rate of 79.6-94.4%. Extraction by the modified technique with fecal samples of over 1 g showed detection of copro-DNA in all samples in both the patent and prepatent periods, and it produced reproducible detection in the addition recovery test using feces from 72 different domestic dogs. As copro-DNA was detected for at least 1 day following deworming with administration of anthelmintic drugs in experimentally infected dogs, diagnostic deworming might be useful for clinical examination. Using the present detection method can provide quick and accurate diagnosis of canine E. multilocularis infection, which with prompt management and treatment of infected dogs can prevent pet owners from becoming infected and prevent echinococcosis from spreading into non-endemic areas.

Synthesis and Antigenicity against Human Sera of a Biotin-Labeled Oligosaccharide Portion of a Glycosphingolipid from the Parasite Echinococcus multilocularis.

Synthesis of a biotinylated analog of the carbohydrate portion of a glycosphingolipid from the parasite Echinococcus multilocularis has been achieved. We synthesized ß-D-Galp-(1→6)-ß-D-Galp-(1→6)-[α-L-Fucp-(1→3)]-ß-D-Galp-(1→R: biotin probe) (1) and compared the antigenicity by an enzyme linked immunosorbent assay (ELISA) with biotinylated trisaccharide α-D-Galp-(1→4)-ß-D-Galp-(1→3)-α-D-Galp-(1→R: biotin probe) (F), which has been shown to have significant antigenicity. Both of the oligosaccharides reacted with sera of alveolar echinococcosis (AE) patients, but showed different reactivity. Among the 60 sera of AE patients, more sera reacted with the linear sequence Galα1→4Galß1→3GalNAcα1→R of oligosaccharide (F) than for branched compound 1. Some sera showed high specificity to one of the compound, indicating that the antibodies in the sera of AE patients differ in their specificity to recognize carbohydrate sequences of glycosphingolipids. Our results demonstrate that both of the biotinylated oligosaccharides 1 and F have good serodiagnostic potential and are complementary to detect infections caused by the parasite Echinococcus multilocularis.

First detection of Echinococcus multilocularis infection in two species of nonhuman primates raised in a zoo: a fatal case in Cercopithecus diana and a strongly suspected case of spontaneous recovery in Macaca nigra.

The causative parasite of alveolar echinococcosis, Echinococcus multilocularis, maintains its life cycle between red foxes (Vulpes vulples, the definitive hosts) and voles (the intermediate hosts) in Hokkaido, Japan. Primates, including humans, and some other mammal species can be infected by the accidental ingestion of eggs in the feces of red foxes. In August 2011, a 6-year-old zoo-raised female Diana monkey (Cercopithecus diana) died from alveolar echinococcosis. E. multilocularis infection was confirmed by histopathological examination and detection of the E. multilocularis DNA by polymerase chain reaction (PCR). A field survey in the zoo showed that fox intrusion was common, and serodiagnosis of various nonhuman primates using western blotting detected a case of a 14-year-old female Celebes crested macaque (Macaca nigra) that was weakly positive for E. multilocularis. Computed tomography revealed only one small calcified lesion (approximately 8mm) in the macaque's liver, and both western blotting and enzyme-linked immunosorbent assay (ELISA) showed a gradual decline of antibody titer. These findings strongly suggest that the animal had recovered spontaneously. Until this study, spontaneous recovery from E. multilocularis infection in a nonhuman primate had never been reported.

Characterization of a surface glycoprotein from Echinococcus multilocularis and its mucosal vaccine potential in dogs.

Alveolar echinococcosis is a refractory disease caused by the metacestode stage of Echinococcus multilocularis. The life cycle of this parasite is maintained primarily between foxes and many species of rodents; thus, dogs are thought to be a minor definitive host except in some endemic areas. However, dogs are highly susceptible to E. multilocularis infection. Because of the close contact between dogs and humans, infection of dogs with this parasite can be an important risk to human health. Therefore, new measures and tools to control and prevent parasite transmission required. Using 2-dimensional electrophoresis followed by western blot (2D-WB) analysis, a large glycoprotein component of protoscoleces was identified based on reactivity to intestinal IgA in dogs experimentally infected with E. multilocularis. This component, designated SRf1, was purified by gel filtration using a Superose 6 column. Glycosylation analysis and immunostaining revealed that SRf1 could be distinguished from Em2, a major mucin-type antigen of E. multilocularis. Dogs (n=6) were immunized intranasally with 500 µg of SRf1 with cholera toxin subunit B by using a spray syringe, and a booster was given orally using an enteric capsule containing 15 mg of the same antigen. As a result, dogs immunized with this antigen showed an 87.6% reduction in worm numbers compared to control dogs (n=5) who received only PBS administration. A weak serum antibody response was observed in SRf1-immunized dogs, but there was no correlation between antibody response and worm number. We demonstrated for the first time that mucosal immunization using SRf1, a glycoprotein component newly isolated from E. multilocularis protoscoleces, induced a protection response to E. multilocularis infection in dogs. Thus, our data indicated that mucosal immunization using surface antigens will be an important tool to facilitate the development of practical vaccines for definitive hosts.

Molecular cloning and characterization of major vault protein of Echinococcus multilocularis.

The cDNA clone coding a major vault protein (MVP)-like protein was derived from Echinococcus multilocularis cysts. MVP is a main component of vault particles, which are the largest cytoplasmic ribonucleoprotein particles in eukaryotic cells. We sequenced and characterized E. multilocularis MVP (EmMVP). The nucleotide sequence of the emmvp cDNA clone was 2607 bp in the full length open reading frame and its deduced amino acid sequence had several signature motifs which were specific to MVP families. Immunoblot analysis with mouse anti-EmMVP antiserum revealed that crude antigens of E. multilocularis included EmMVP protein. Furthermore, our results showed that the expression of EmMVP protein in an Sf9 insect cell line using a baculovirus vector directed the formation of particles that shared similar biochemical characteristics with other vault proteins and the distinct vault-like morphology when negatively stained and examined by electron microscopy.

Synthesis, antigenicity against human sera and structure-activity relationships of carbohydrate moieties from Toxocara larvae and their analogues.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the Galß1-3GalNAc core of the TES-glycoprotein antigen obtained from larvae of the parasite Toxocara and their analogues have been accomplished. Trisaccharides Fuc2Meα1-2Gal4Meß1-3GalNAcα1-OR (A), Fucα1-2Gal4Meß1-3GalNAcα1-OR (B), Fuc2Meα1-2Galß1-3GalNAcα1-OR (C), Fucα1-2Galß1-3GalNAcα1-OR (D) and a disaccharide Fuc2Meα1-2Gal4Meß1-OR (E) (R = biotinylated probe) were synthesized by block synthesis using 5-(methoxycarbonyl)pentyl-2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1-->3)-2-azide-4-O-benzyl-2-deoxy-α-D-galactopyranoside as a common glycosyl acceptor. We examined the antigenicity of these five oligosaccharides by enzyme linked immunosorbent assay (ELISA). Our results demonstrate that the O-methyl groups in these oligosaccharides are important for their antigenicity and the biotinylated oligosaccharides A, B, C and E have high serodiagnostic potential to detect infections caused by Toxocara larvae.

Galα1-4Galß1-3GalNAc is the dominant epitope of Em2 antigen, the mucin-type glycoprotein from Echinococcus multilocularis.

The larval stage of Echinococcus multilocularis causes alveolar echinococcosis in human. In serodiagnosis of alveolar echinococcosis, specific reactions have been noted not only against protein antigens but also carbohydrates. With regard to protein antigens, the recent development of recombinant antigens has contributed to an improvement in serodiagnostic examination. On the contrary, the preparation of carbohydrate antigen still depends on extraction from crude antigens, and isolation is usually accompanied with difficulty; consequently, it is rare to examine individual antigenicity of carbohydrates. However, parasitic helminths express various antigenic carbohydrates. In the case of Echinococcus granulosus, antigenic glycoproteins of the laminated layer have been reported. Furthermore, the laminated layer of E. multilocularis contains Em2 antigen which is a famous mucin-type glycoprotein and which seems to play an important role in metacestode survival mechanisms within the immunologically reacting host; nevertheless, the anomeric configurations and the individual antigenicity of Em2 O-glycans have not been confirmed so far. Under these circumstances, we introduced a chemical synthesis to get pure oligosaccharides in order to assess diagnostic performance. In our previous study, 11 oligosaccharides have already been prepared by stereocontrolled syntheses. Among them, three synthetic oligosaccharides showed antigenicity. Our aim is to investigate correct sequence and serodiagnostic potential of the dominant epitope of Em2. This study provided important diagnostic information: (1) the trisaccharide Galα1-4Galß1-3GalNAc sequence is the dominant epitope of Em2 (sensitivity 95.0 %), (2) Trematoda expresses carbohydrates with the similar trisaccharide sequence, and (3) the terminal Galα1-4Gal sequence is a candidate for the widely common epitope that accounts for the cross-reaction.

Synthesis of the carbohydrate moiety from the parasite Echinococcus multilocularis and their antigenicity against human sera.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions with a Galß1-3GalNAc core of the Em2 glycoprotein antigen obtained from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Galß1-3(GlcNAcß1-6)GalNAcα1-R (G), tetrasaccharide Galß1-3(Galß1-4GlcNAcß1-6)GalNAcα1-R (J) and pentasaccharide Galß1-3(Galα1-4Galß1-4GlcNAcß1-6)GalNAcα1-R (K) (R=biotinylated probe) were synthesized by block synthesis by the use of 5-(methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1→3)-2-azido-4-O-benzyl-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. Moreover, linear trisaccharide Galα1-4Galß1-3GalNAcα1-R (H) and branched tetrasaccharide Galα1-4Galß1-3(GlcNAcß1-6)GalNAcα1-R (I) were synthesized by stepwise condensation. We examined the antigenicity of these five oligosaccharides by an enzyme linked immunosorbent assay (ELISA). Our results demonstrate that biotinylated oligosaccharides H, I and K show good serodiagnostic potential to detect infections caused by the parasite E. multilocularis. Among them the linear sequence Galα1-4Galß1-3GalNAcα1-R in oligosaccharide (H) appears to show the highest sensitivity (95%). Moreover, our study clarified the dominant carbohydrate epitope of Em2 antigen.

Echinococcus multilocularis: purification and characterization of glycoprotein antigens with serodiagnostic potential for canine infection.

We show that a conventionally purified glycoprotein component of Echinococcus multilocularis protoscolex, designated as Emgp-89, may be useful as a serodiagnostic antigen for detecting E. multilocularis infection in dogs domesticated in endemic areas. Emgp-89 was obtained from the parasite material by a simple procedure using Con A-agarose and subsequent gel filtration chromatography. The purified fraction showed a molecular weight of >4000kDa upon gel filtration and reacted with a series of lectins that specifically bind to mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Subsequently, serodiagnostic performance of Emgp-89 was evaluated through enzyme-linked immunosorbent assays (ELISAs) by using sera from normal, domestic dogs and dogs infected with other helminths. Emgp-89 positively reacted with all 16 serum samples from E. multilocularis-infected dogs, thus showing that this antigen is highly sensitive. On the other hand, the specificity of Emgp-89-based ELISA, determined using 41 serum samples from dogs infected with other helminths, was relatively low (83%). As an attempt to improve the specificity of Emgp-89-based ELISA, we pretreated Emgp-89 with proteinase K or sodium periodate, expecting that these treatments would enable discrimination of true positives from false positives. The ELISA value increased after treatment with sodium periodate in most false-positive samples, whereas significant decreases were observed in sera from all dogs infected with E. multilocularis. Further evaluation of this antigen should be performed using sera from dogs infected with closely-related parasites, including taeniid cestodes, which are expected to prove that this serodiagnostic system is sufficiently specific for clinical and field applications.

Infection of humans and animals with Echinococcus granulosus (G1 and G3 strains) and E. ortleppi in Southern Brazil.

The Rio Grande do Sul state, in Southern Brazil, is one of the foci of human cystic echinococcosis (CE). The sheep strain (G1) of Echinococcus granulosus and Echinococcus ortleppi (also known as cattle strain G5) have been reported before to infect livestock. However, up to the present, no molecular data are available on isolates of the E. granulosus complex from humans and dogs. The present study analyzed hydatid cysts from 6 CE patients and adult worms from 12 dogs. Sequencing of the mitochondrial cox1 and 12S rRNA genes detected the E. granulosus G1 genotype from four human cases, the G3 genotype (or buffalo strain) from one human case and E. ortleppi from another human case, respectively. Ten of the twelve dogs were found infected with the G1 genotype, and one dog each harbored worms of the G3 genotype and E. ortleppi. Obvious morphological differences were recognized between the G1 and E. ortleppi adult worms from dogs in this region. The buffalo strain (G3) is for the first time reported from South America.

Serological reactivity of patients with Echinococcus infections (E. granulosus, E. vogeli, and E. multilocularis) against three antigen B subunits.

In serodiagnosis of cystic echinococcosis (CE) by Echinococcus granulosus infection, antigen B (AgB) has been utilized worldwide. However, it is known that about 40% of sera with alveolar echinococcosis (AE) by Echinococcus multilocularis infection recognize AgB. Furthermore, cross-reaction against AgB was also reported in sera from polycystic echinococcosis (PE) patients with Echinococcus vogeli infection. These findings indicate that AgB is widely common to the genus Echinococcus. On the other hand, AgB has several subunits, which are composed of the smallest 8-kDa subunit. In this study, reactivities of patient sera with three kinds of Echinococcus infections (CE, PE, and AE) were compared simultaneously under the same condition against three subunits of AgB (8, 16, and 24 kDa). Many articles have referred the fundamental 8- kDa subunit as a diagnostic antigen for CE. However, the reactivity for the 8-kDa subunit of the CE patient was not so high (47.7%) in this study. Furthermore, there are many cases in which serum of patients with PE or AE also recognizes this subunit (66.7% in PE; 45.9% in AE). AgB is effective for the detection of the genus Echinococcus infections, but it does not have high species specificity. Therefore, we need to pay attention to cross-reaction in serodiagnosis of CE in areas where plural species coexist.

Japanese monkey (Macaca fuscata) with alveolar echinococcosis after treatment with albendazole for 10 years: serodiagnosis and determination of albendazole metabolites.

In 1997, an outbreak of alveolar echinococcosis in Japanese monkeys (Macaca fuscata) occurred in a zoo in Hokkaido, Japan. Twelve infected monkeys from a colony (n = 57) were diagnosed serologically by Western blotting, and ultrasonography showed the presence of tumor-like tissue in the livers of nine monkeys. The 12 infected monkeys have been treated with albendazole for 10 years without surgical resection. Ten of these monkeys have died so far; diagnoses were confirmed histopathologically through autopsy. Two of these monkeys are still alive. Recently, a significant difference between the two living monkeys was recognized. A difference in curative effect was demonstrated between the two living monkeys by radiography, contrast enhanced computed tomography, and contrast ultrasound. One showed metastasis to various organs, and the other appeared to be almost cured, as demonstrated by size reduction and calcification of the lesion after albendazole treatment for 10 years. This time, serological reexamination was performed to corroborate this apparent difference. The serological tests supported the preliminary imaging findings. In addition, the presence of albendazole metabolites in sera was confirmed by high-performance liquid chromatography. In this study, it was demonstrated that tests which have been used in human cases were also effective for diagnosing alveolar echinococcosis and for assessing curative effects in nonhuman primates such as M. fuscata.

Significance of imaging features of alveolar echinococcosis in studies on nonhuman primates.

In this study, we report the imaging findings in two Japanese monkeys (Macaca fuscata) diagnosed with alveolar echinococcosis. Both monkeys were treated with albendazole for 10 years, without surgery. Radiography, computed tomography, and contrast-enhanced ultrasonography were performed under general anesthesia. This is the first report on contrast-enhanced ultrasonographic imaging for alveolar echinococcosis wherein perflubutane was used as the contrast medium. The findings of the imaging analyses were similar to those reported for alveolar echinococcosis in humans, such as snowflake sign and worm-eaten sign. In addition, the serology correlated well with the imaging data in these two monkeys. Therefore, we propose that the imaging findings of alveolar echinococcosis in nonhuman primates may be used to accumulate data on this condition in human alveolar echinococcosis.

Synthetic studies on the carbohydrate moiety of the antigen from the parasite Echinococcus multilocularis.

Stereocontrolled syntheses of branched tri-, tetra-, and pentasaccharides displaying a Gal beta 1-->3GalNAc core in the glycan portion of the glycoprotein antigen from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc alpha 1-OR (A), tetrasaccharide Gal beta 1-->3(Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (D), and pentasaccharides Gal beta 1-->3(Gal beta 1-->4Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (E) and Gal beta1-->3(Gal alpha 1-->4Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (F) (R = 2-(trimethylsilyl)ethyl) were synthesized by block synthesis. The disaccharide 2-(trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-azido-4-O-benzyl-2-deoxy-alpha-d-galactopyranoside served as a common glycosyl acceptor in the synthesis of the branched oligosaccharides. Moreover, linear trisaccharide Gal beta 1-->4Gal beta 1-->3GalNAc alpha 1-OR (B) and branched tetrasaccharide Gal beta 1-->4Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc alpha 1-OR (C) were synthesized by stepwise condensation.

Mouse monoclonal immunoglobulin E antibodies specific for the microsporidian Encephalitozoon cuniculi polar tube protein 1.

The microsporidian Encephalitozoon cuniculi is a spore-forming, obligate, intracellular parasitic pathogen with a unique organelle called a polar tube, the extrusion of which is essential for invading a host cell. The polar tube consists of three proteins: polar tube protein 1 (PTP1), PTP2, and PTP3. We established three mouse monoclonal antibodies (MAb1, MAb2, and MAb4) against E. cuniculi PTP1. An enzyme-linked immunosorbent assay (ELISA) indicated that all three MAbs reacted with the outer surface of extruded polar tubes and that they also strongly bound to an intracellular antigen in infected host cells. Two-dimensional (2-D) immunoblot analysis showed that MAb1 and MAb2 recognized PTP1 spots at 52 kDa and some spotty smears at molecular weights of less than 50 kDa, whereas MAb4 recognized only PTP1 spots at 52 kDa. Interestingly, all three MAbs were of the immunoglobulin (Ig) E class, suggesting that, in addition to the highly immunogenic or antigenic nature, the PTP1 antigen may have the potential to induce specific IgE antibody production in mice. These antibodies may be useful in the study of allergenic PTP1 as well in the purification and detection of the PTP1 antigen.