Gut microbiota differences induced by Toxoplasma gondii seropositivity in stray cats in South Korea.

T. gondii is a highly prevalent parasite worldwide, with cats serving as its final host. However, few studies have investigated the impact of T. gondii infection on cat gut microbiota. Therefore, this study examined the influence of T. gondii infection on the gut microbiota of stray cats and identified potential pathogens in their feces. This study examined T. gondii infection through blood of stray cats and the influence of microbiota in their feces using 16S rRNA gene amplicon sequencing. The results revealed significant differences in gut microbiota composition and diversity between the T. gondii seropositive and seronegative groups. Seropositive samples displayed a lower number of operational taxonomic units and reduced Shannon index than the seronegative samples. The seropositive and seronegative groups exhibited enrichment of taxa, including Escherichia and Enterobacteriaceae and Collinsella, Bifidobacterium, and Roseburia, respectively. Furthermore, potential pathogen species, including Campylobacter, Escherichia, and Streptococcus, were identified in the fecal samples. These findings suggest that T. gondii infection significantly impacts gut microbiota composition and diversity in stray cats. Additionally, an increased potential pathogen load, represented by Escherichia spp., was observed. These results underscore the importance of monitoring the prevalence of zoonotic pathogens in stray cats, as they can serve as reservoirs for zoonotic diseases.

Molecular characteristics and induction profiles of hypoxia-inducible factor-1α and other basic helix-loop-helix and Per-Arnt-Sim domain-containing proteins identified in a carcinogenic liver fluke Clonorchis sinensis.

Clonorchis sinensis (C. sinensis), a trematode parasite that invades the hypoxic hepatobiliary tract of vertebrate hosts requires a considerable amount of oxygen for its sexual reproduction and energy metabolism. However, little is known regarding the molecular mechanism of C. sinensis involved in the adaptation to the hypoxic environments. In this study, we investigated the molecular structures and induction patterns of hypoxia-inducible factor-1α (HIF-1α) and other basic helix-loop-helix and Per-Arnt-Sim (bHLH-PAS) domain-containing proteins such as HIF-1ß, single-minded protein and aryl hydrocarbon receptor, which might prompt adaptive response to hypoxia, in C. sinensis. These proteins possessed various bHLH-PAS family-specific domains. Expression of C. sinensis HIF-1α (CsHIF-1α) was highly induced in worms which were either exposed to a hypoxic condition or co-incubated with human cholangiocytes. In addition to oxygen, nitric oxide and nitrite affected the CsHIF-1α expression depending on the surrounding oxygen concentration. Treatment using a prolyl hydroxylase-domain protein inhibitor under 20%-oxygen condition resulted in an increase in the CsHIF-1α level. Conversely, the other bHLH-PAS genes were less responsive to these exogenous stimuli. We suggest that nitrite and nitric oxide, as well as oxygen, coordinately involve in the regulation of HIF-1α expression to adapt to the hypoxic host environments in C. sinensis.

Inactivation of Paragonimus westermani metacercariae in soy sauce-marinated and frozen freshwater crabs.

Soy sauce-marinated freshwater crabs (Eriocheir japonicus) are a source of human paragonimiasis. The viability of Paragonimus westermani metacercariae (PwMc) in marinated crabs was investigated in an experimental setting. The PwMc collected from freshwater crayfish were inoculated into freshwater crabs, which were then frozen or marinated in soy sauce. All PwMc in the freshwater crabs were inactivated after freezing for 48 h at -20 °C and after freezing for 12 h at -40 °C. After marinating for 32 days, the survival rate of PwMc in 5% NaCl soy sauce was 50%, in 7.5% NaCl soy sauce it was 33.3%, and in 10.0% NaCl soy sauce it was 31.3%. When marinated for 64 days, all PwMc were inactivated in all experimental groups. These results revealed that freezing and soy sauce marination were detrimental to the survival of PwMc in freshwater crabs. Specifically, freezing crabs for more than 48 h or soaking them in soy sauce containing at least 5.0% NaCl for 64 days can inactivate PwMc. These results can inform the production of the traditional Korean soy sauce-marinated freshwater crabs known as gejang.

Isolation and Characterization of Vaccine Candidate Genes Including CSP and MSP1 in Plasmodium yoelii.

Malaria is an infectious disease affecting humans, which is transmitted by the bite of Anopheles mosquitoes harboring sporozoites of parasitic protozoans belonging to the genus Plasmodium. Despite past achievements to control the protozoan disease, malaria still remains a significant health threat up to now. In this study, we cloned and characterized the full-unit Plasmodium yoelii genes encoding merozoite surface protein 1 (MSP1), circumsporozoite protein (CSP), and Duffy-binding protein (DBP), each of which can be applied for investigations to obtain potent protective vaccines in the rodent malaria model, due to their specific expression patterns during the parasite life cycle. Recombinant fragments corresponding to the middle and C-terminal regions of PyMSP1 and PyCSP, respectively, displayed strong reactivity against P. yoelii-infected mice sera. Specific native antigens invoking strong humoral immune response during the primary and secondary infections of P. yoelii were also abundantly detected in experimental ICR mice. The low or negligible parasitemia observed in the secondary infected mice was likely to result from the neutralizing action of the protective antibodies. Identification of these antigenic proteins might provide the necessary information and means to characterize additional vaccine candidate antigens, selected solely on their ability to produce the protective antibodies.

Recent Incidence of Paragonimus westermani Metacercariae in Freshwater Crayfish, Cambaroides similis, from Two Enzootic Sites in Jeollanam-do, Korea.

An epidemiological study was performed to know the recent infection status of Paragonimus westermani metacercariae (PwMc) in freshwater crayfish, Cambaroides similis, from 2 streams in Jeollanam-do, Republic of Korea. Crayfish were collected from creeks in Bogil-do (Island), Wando-gun, and in a creek near Daeheung Temple in Haenam-gun. The infection rate of crayfish with PwMc in Bogil-do was 89.8%, and the metacercarial burden was 37 PwMc per the infected crayfish. Crayfish in a creek near Daeheung Temple were larger and twice heavier than those in Bogil-do. Of them, 96.5% were infected with PwMc. An average of 140 metacercariae was found in the infected crayfish, almost quadruple to those of Bogil-do. There was a strong correlation between the number of PwMc and body weight of the crayfish. These results suggest that P. westermani metacercariae are still prevalent in crayfish of the 2 regions in Jeollanam-do, Korea.

Spatiotemporal Expression Patterns and Antibody Reactivity of Taeniidae Endophilin B1.

Larval Taeniidae, such as metacestodes of Taenia solium, Echinococcus granulosus, and Echinococcus multilocularis, produce chronic and fatal helminthic diseases. Proper identification of these zoonotic cestodiases is often challenging and is hampered in some clinical settings. Endophilin B1 plays critical roles in the maintenance of membrane contours and endocytosis. We isolated proteins homologous to endophilin B1 from T. solium, Taenia saginata, and Taenia asiatica The three Taeniidae endophilin B1 proteins shared 92.9 to 96.6% sequence identity. They harbored a Bin1/amphiphysin/Rvs (BAR) domain and residues for a dimeric interface but lacked a SRC homology 3 (SH3) domain. Endophilin B1 showed a unique immunological profile and was abundantly expressed in the tegumental syncytium of Taeniidae metacestodes and adults. Bacterially expressed recombinant T. solium endophilin B1 (rTsMEndoB1) demonstrated a sensitivity of 79.7% (345/433 cases) for serodiagnosis of larval Taeniidae infections. The protein showed strong immune recognition patterns against sera from patients with chronic neurocysticercosis, cystic echinococcosis, or advanced-stage alveolar echinococcosis. Adult Taeniidae infections exhibited moderate degrees of positive antibody responses (65.7% [23/35 samples]). rTsMEndoB1 showed some cross-reactivity with sera from patients infected with Diphyllobothriidae (23.6% [25/106 samples]) but not with sera from patients with other parasitic diseases or normal controls. The specificity was 91.7% (256/301 samples). The positive and negative predictive values were 93.6% and 73.4%, respectively. Our results demonstrate that Taeniidae endophilin B1 may be involved in the control of membrane dynamics, thus contributing to shaping and maintaining the tegumental curvature. rTsMEndoB1 may be useful for large-scale screening, as well as for individual diagnosis and follow-up surveillance of Taeniidae infections.

An Echinococcus multilocularis Antigen B3 Proteoform That Shows Specific Antibody Responses to Active-Stage Alveolar Echinococcosis.

Alveolar echinococcosis (AE), caused by the Echinococcus multilocularis metacestode, represents one of the most frequently fatal zoonoses. Early diagnosis significantly reduces morbidity and mortality associated with AE. Diagnosis of AE largely depends on a combination of imaging and serological tests due to its minimal clinical manifestations. Several antigens derived from the whole worm and protoscolex have been targeted for AE serodiagnosis, while the antigenic properties of E. multilocularis hydatid fluid (EmHF) are unclear. We observed two AE-specific 6- and 8-kDa antigen proteoforms through an immunoproteome array of the EmHF. We identified these proteins as representing an E. multilocularis antigen B3 (EmAgB3) isoform, and the proteins were shown to be encoded by the same gene. We cloned the gene and expressed the recombinant EmAgB3 protein (rEmAgB3) in Escherichia coli. rEmAgB3 exhibited sensitivity of 90.9% (80/88 cases) and specificity of 98.5% (597/606 samples) by immunoblotting. The positive and negative predictive values were 89.9% and 98.6%, respectively. The protein did not show antibody responses to 33 AE sera collected during posttreatment follow-up monitoring. Mouse sera experimentally infected with AE protoscoleces began to demonstrate specific antibody responses to native and recombinant EmAgB3 6 months after infection. At that stage, fully mature metacestode vesicles that harbored the brood capsule, primary cell, and protoscolex were observed within an AE mass(es). The response declined along with worm degeneration. Our results demonstrate that the immune responses to this EmAgB3 isoform were highly correlated with worm viability accompanied with AE progression. rEmAgB3 is a promising biomarker for serological assessment of AE patients.

Egg-specific expression of protein with DNA methyltransferase activity in the biocarcinogenic liver fluke Clonorchis sinensis.

Despite recent reports regarding the biology of cytosine methylation in Schistosoma mansoni, the impact of the regulatory machinery remains unclear in diverse platyhelminthes. This ambiguity is reinforced by discoveries of DNA methyltransferase 2 (DNMT2)-only organisms and the substrate specificity of DNMT2 preferential to RNA molecules. Here, we characterized a novel DNA methyltransferase, named CsDNMT2, in a liver fluke Clonorchis sinensis. The protein exhibited structural properties conserved in other members of the DNMT2 family. The native and recombinant CsDNMT2 exhibited considerable enzymatic activity on DNA. The spatiotemporal expression of CsDNMT2 mirrored that of 5-methylcytosine (5 mC), both of which were elevated in the C. sinensis eggs. However, CsDNMT2 and 5 mC were marginally detected in other histological regions of C. sinensis adults including ovaries and seminal receptacle. The methylation site seemed not related to genomic loci occupied by progenies of an active long-terminal-repeat retrotransposon. Taken together, our data strongly suggest that C. sinensis has preserved the functional DNA methylation machinery and that DNMT2 acts as a genuine alternative to DNMT1/DNMT3 to methylate DNA in the DNMT2-only organism. The epigenetic regulation would target functional genes primarily involved in the formation and/or maturation of eggs, rather than retrotransposons.

Taenia solium metacestode fasciclin-like protein is reactive with sera of chronic neurocysticercosis.

OBJECTIVES: Neurocysticercosis (NC), an infection of the central nervous system with Taenia solium metacestodes (TsM), invokes a formidable neurological disease. A bundle of antigens is applicable for serodiagnosis of active cases, while they demonstrate fairly low reactivity against sera of chronic NC. Identification of sensitive biomarkers for chronic NC is critical for appropriate management of patients. METHODS: Proteome analysis revealed several isoforms of 65- and 83-kDa TsM fasciclin-like proteins (TsMFas) to be highly reactive with sera of chronic NC. A cDNA encoding one of the 83-kDa TsMFas (TsMFas1) was isolated from a cDNA library. We expressed a recombinant protein (rTsMFas1) and evaluated its diagnostic potential employing sera from chronic NC (n = 80), tissue-invasive cestodiases (n = 169) and trematodiases (n = 80) and those of normal controls (n = 50). RESULTS: Secretory TsMFas1 was composed of 766 amino acid polypeptide and harboured fasciclin and fasciclin-superfamily domains. The protein was constitutively expressed in metacestode and adult stages, with preferential locality in the scolex. Bacterially expressed rTsMFas1 exhibited 78.8% sensitivity (63/80 cases) and 93% specificity (278/299 samples) in diagnosing chronic NC. Some cross-reactivity was observed with sera of cystic echinococcosis (10/56, 17.8%) and sparganosis (4/50, 8%). Positive and negative predictive values were 75% and 95.5%, respectively. CONCLUSION: TsM fasciclin-like protein may be useful for differential diagnosis of chronic NC in clinical settings, especially where both NC and other infectious cerebral granulomatoses are prevalent.

Metagonimus yokogawai: a 100-kDa somatic antigen commonly reacting with other trematodes.

This study was undertaken to characterize the properties of a 100 kDa somatic antigen from Metagonimus yokogawai. Monoclonal antibodies (mAbs) were produced against this 100 kDa antigen, and their immunoreactivity was assessed by western blot analysis with patients' sera. The mAbs against the 100 kDa antigen commonly reacted with various kinds of trematode antigens, including intestinal (Gymnophalloides seoi), lung (Paragonimus westermani), and liver flukes (Clonorchis sinensis and Fasciola hepatica). However, this mAb showed no cross-reactions with other helminth parasites, including nematodes and cestodes. To determine the topographic distribution of the 100 kDa antigen in worm sections, indirect immunoperoxidase staining was performed. A strong positive reaction was observed in the tegumental and subtegumental layers of adult M. yokogawai and C. sinensis. The results showed that the 100 kDa somatic protein of M. yokogawai is a common antigen which recognizes a target epitope present over the tegumental layer of different trematode species.

Functional changes in regulatory T cells during an experimental infection with sparganum (plerocercofid of Spirometra mansoni).

Regulatory T (Treg) cells are important in the regulation of immune response, but the exact regulation of Treg-cell function in vivo is still not well known. In the present study, we investigated the functional activity of CD4(+) CD25(+) Treg cells as well as the frequency and number of CD4(+) CD25(+) FoxP3(+) Treg cells in the spleens of experimentally infected mice with a tissue-migrating parasite, sparganum (plerocercoid of Spirometra mansoni) for 3 weeks. The results demonstrated fluctuations in the Treg-cell function during the parasite infection, being up-regulated at day 3, down-regulated until day 14, and thereafter up-regulated again at day 21. We also investigated the cytokine-producing capability of the splenocytes to study the pattern of immune response of the mice to the parasite. The results showed decreased capabilities of interleukin-2 (IL-2), interferon-γ (IFN-γ) and IL-17α production, whereas IL-4-producing and IL-10-producing capabilities were increased along with the parasitic infection. Meanwhile, IL-6-producing capability was increased to reach a peak at week 2, and thereafter was decreased to the baseline level. As a regulatory mechanism, we found that Treg-cell function was attenuated in the presence of the crude extracts of sparganum, but was enhanced in the presence of the excretory-secretory products, suggesting that sparganum products were involved in the triggering and regulation of immune response in the acute and chronic phases, respectively. Results show that Treg cells are central in the immune homeostasis in vivo that is maintained by host-parasite interactions during the parasitic infection.

Electron microscopy of the separated outer tegument of the sparganum and its antigenicity.

The author reported previously on separation of the outer tegument of the spargana (plerocercoids of Spirometra mansoni) using high concentration of urea solution. To determine which layer of the tegument is separated by this method, an electron microscopic analysis has been processed in this study. It was confirmed that the basement layer of the tegument is separated from the parenchyme of the sparganum. In addition, the antigenicity of the separated outer tegument against the human sparganosis patient sera was evaluated. Numerous antigenic proteins, including 16 and 55 kDa proteins, were noticed in the separated tegument; however, there were no diagnostic 31/36 kDa molecules in this tegument. The molecules reactive with the patient sera in the tegument are to be characterized in future studies.

Four human cases of Diphyllobothrium latum infection.

Diphyllobothrium latum infections in 4 young Korean men detected from 2008 to 2012 are presented. Three were diagnosed based on spontaneously discharged strobila of the adult worm in their feces, and 1 case was diagnosed by finding the worm at colonoscopy examination in a local clinic. The morphologic characteristics of the gravid proglottid and eggs were consistent with D. latum. All patients were treated with praziquantel 15 mg/kg, and follow-up stool examinations were done at 2 months after the medication. The main clinical complaints were intermittent gastrointestinal troubles such as indigestion, abdominal distension, and spontaneous discharge of tapeworm's segments in their feces. The most probable source of infection was the flesh of salmon or trout according to a patient's past history. These are the 45th to 48th recorded cases diagnosed by the adult worm in the Republic of Korea since 1971.

Experimental murine fascioliasis derives early immune suppression with increased levels of TGF-ß and IL-4.

In fascioliasis, T-helper 2 (Th2) responses predominate, while little is known regarding early immune phenomenon. We herein analyzed early immunophenotype changes of BALB/c, C57BL/6, and C3H/He mice experimentally infected with 5 Fasciola hepatica metacercariae. A remarkable expansion of CD19(+) B cells was observed as early as week 1 post-infection while CD4(+)/CD8(+) T cells were down-regulated. Accumulation of Mac1(+) cells with time after infection correlated well with splenomegaly of all mice strains tested. The expression of tumor necrosis factor (TNF)-α mRNA in splenocytes significantly decreased while that of IL-4 up-regulated. IL-1ß expression was down-modulated in BALB/c and C57BL/6 mice, but not in C3H/He. Serum levels of transforming growth factor (TGF)-ß were considerably elevated in all mice during 3 weeks of infection period. These collective results suggest that experimental murine fascioliasis might derive immune suppression with elevated levels of TGF-ß and IL-4 during the early stages of infection.

Expression of TNF-α and IL-1ß in splenic dendritic cells and their serum levels in mouse sparganosis.

Sparganosis is a tissue invading helminthiasis infecting intermediate hosts, including humans. Strong immune responses are expected to occur in early phases of infection. Thus, we investigated cytokine expressions in splenic dendritic cells and in sera after experimental infection of mice. In splenic dendritic cells, TNF-α and IL-1ß expression peaked at week 1 and week 3 post-infection (PI), respectively, and also early phase (week 2 PI) depressed cytokine expression was noticed. Serum IL-1ß concentration increased significantly at week 2 PI and peaked at week 6 PI, and that of TNF-α peaked at week 6 PI. These results showed that pro-inflammatory cytokines, TNF-α and IL-1ß, are chronologically regulated in mouse sparganosis.

Pro-inflammatory cytokine expression of spleen dendritic cells in mouse toxoplasmosis.

Dendritic cells have been known as a member of strong innate immune cells against infectious organelles. In this study, we evaluated the cytokine expression of splenic dendritic cells in chronic mouse toxoplasmosis by tissue cyst-forming Me49 strain and demonstrated the distribution of lymphoid dendritic cells by fluorescence-activated cell sorter (FACS). Pro-inflammatory cytokines, such as IL-1α, IL-1ß, IL-6, and IL-10 increased rapidly at week 1 post-infection (PI) and peaked at week 3 PI. Serum IL-10 level followed the similar patterns. FACS analysis showed that the number of CD8α(+)/CD11c(+) splenic dendritic cells increased at week 1 and peaked at week 3 PI. In conclusion, mouse splenic dendritic cells showed early and rapid cytokine changes and may have important protective roles in early phases of murine toxoplasmosis.

Production of polyclonal antibodies against the tegument of sparganum (plerocercoid of Spirometra mansoni) and its immunolocalization.

In a previous study, the author developed a method for separation of the tegument of spargana (plerocercoids of Spirometra mansoni) from the parenchyme using urea. The present study, as a next step, was performed to evaluate which molecules are present in the outer tegument. Two major proteins, 180 and 200 kDa, are present in the tegument and we could make polyclonal antibodies against these molecules. Their immunolocalization was processed and the outermost layer of the spargana showed strong positive staining. Conclusively, we could confirm that the 180 and 200 kDa molecules might be tightly bound membrane proteins in the tegument of spargana.

Separation of the syncytial layer of spargana using urea.

The tegument of tapeworms is known to be composed of an outer syncytial cytoplasm layer which includes microtriches and cytoplasmic organelles (= syncytial layer), and a parenchymatous cytoplasm layer that contains subtegumental cell nuclei (= subtegumental layer) and organelles. In the present study, separation of the syncytial layer of the sparganum, the plerocercoid stage of Spirometra mansoni, was tried using urea as the chemical reagent. Histological sections were prepared to visualize the status of separation after staining with hematoxylin and eosin. The results showed that the syncytial layer of the sparganum tegument which includes microtriches and cytoplasmic organelles were successfully separated from the parenchyma using 3 M urea.

On-demand route discovery in a unicast manner.

While having high bandwidth-efficiency, the ad-hoc on-demand distance vector (AODV) routing protocol suffers from high signaling overhead due to route request (RREQ) messages flooding, especially when the node density and the number of connections are increased. In order to resolve this broadcast storm problem of the AODV in a high node density mobile ad-hoc network, we propose a geographical on-demand route discovery scheme. Assuming a known location of the destination, the RREQ of the proposed routing protocol is propagated in a unicast manner by employing a novel parsing mechanism for possible duplicate RREQs. The routing overhead of the proposed routing protocol is greatly robust to the node density change. We derive the node density required for the proposed routing protocol to keep the same connectivity as the AODV under the circumstance where the nodes are uniformly distributed. In addition, we present an imaginary destination consideration method to incorporate the uncertainty of the destination's location due to mobility. Computer simulations show that the proposed scheme enables the RREQ propagation to cover 95% of the one-hop communication area centered at the originally known location of the destination without sacrificing the unicast feature.

Clonorchis sinensis omega-class glutathione transferases play major roles in the protection of the reproductive system during maturation and the response to oxidative stress.

BACKGROUND: Clonorchis sinensis causes a major food-borne helminthic infection. This species locates in mammalian hepatobiliary ducts, where oxidative stressors and hydrophobic substances are profuse. To adapt to the hostile micromilieu and to ensure its long-term survival, the parasite continuously produces a diverse repertoire of antioxidant enzymes including several species of glutathione transferases (GSTs). Helminth GSTs play pertinent roles during sequestration of harmful xenobiotics since most helminths lack the cytochrome P-450 detoxifying enzyme. METHODS: We isolated and analyzed the biochemical properties of two omega-class GSTs of C. sinensis (CsGSTo1 and CsGSTo2). We observed spatiotemporal expression patterns in accordance with the maturation of the worm's reproductive system. Possible biological protective roles of CsGSTos in these organs under oxidative stress were investigated. RESULTS: The full-length cDNAs of CsGSTo1 and 2 constituted 965 bp and 1,061 bp with open reading frames of 737 bp (246 amino acids) and 669 bp (223 amino acids). They harbored characteristic N-terminal thioredoxin-like and C-terminal α-helical domains. A cysteine residue, which constituted omega-class specific active site, and the glutathione-binding amino acids, were recognized in appropriate positions. They shared 44 % sequence identity with each other and 14.8-44.8 % with orthologues/homologues from other organisms. Bacterially expressed recombinant proteins (rCsGSTo1 and 2) exhibited dehydroascorbate reductase (DHAR) and thioltransferase activities. DHAR activity was higher than thioltransferase activity. They showed weak canonical GST activity toward 1-chloro-2,4-dinitrobenzene. S-hexylglutathione potently and competitively inhibited the active-site at nanomolar concentrations (0.63 and 0.58 nM for rCsGSTo1 and 2). Interestingly, rCsGSTos exhibited high enzyme activity toward mu- and theta-class GST specific substrate, 4-nitrobenzyl chloride. Expression of CsGSTo transcripts and proteins increased beginning in 2-week-old juveniles and reached their highest levels in 4-week-old adults. The proteins were mainly expressed in the elements of the reproductive system, such as vitelline follicles, testes, seminal receptacle, sperm and eggs. Oxidative stressors induced upregulated expression of CsGSTos in these organs. Regardless of oxidative stresses, CsGSTos continued to be highly expressed in eggs. CsGSTo1 or 2 overexpressing bacteria demonstrated high resistance under oxidative killing. CONCLUSIONS: CsGSTos might be critically involved in protection of the reproductive system during maturation of C. sinensis worms and in response to oxidative conditions, thereby contributing to maintenance of parasite fecundity.