Crude Worm Research Articles

Molecular and biological characterization of transforming growth factor-β homolog derived from Trichinella spiralis

The cytokine homologs, particularly transforming growth factor (TGF)-β, is a crucial immunomodulatory molecule and involved in growth and developmental processes in several helminths. In this study, the basic properties and functions of T. spiralis TGF-β homolog 2 (TsTGH2) were characterized using bioinformatics and molecular biology approaches. Bioinformatics analyses indicated that TsTGH2 belongs to the TGF-β subfamily. Recombinant TsTGH2 (rTsTGH2) expressed in Escherichia coli was used to produce a polyclonal antibody (pAb) in mice. Western blot and immunolocalization using pAb detected native TsTGH2 in crude worm antigens from muscle larvae and adults, showing it was mainly localized in the body wall muscles and the epithelia of the ovary and uterus. To assess the interplay between TsTGH2 and the human TGF-β signaling pathway, rTsTGH2 produced in a HEK293T cell was incubated with the SBE luciferase-HEK293 cell. The result indicated a significant increase in luciferase activity after treatment with rTsTGH2 compared to untreated control (p < 0.05). In conclusion, these findings are the first to characterize the basic properties and functions of TGF-β homologs in T. spiralis, demonstrating their interaction with the human TGF-β receptor. Further investigation is required to identify and optimize an appropriate expression system or conditions for TsTGH2. Additionally, studies are needed to clarify the specific role of native TsTGH2 in parasite development and host immunomodulation.

Cystatins from the Human Liver Fluke Opisthorchis viverrini: Molecular Characterization and Functional Analysis.

A high incidence of cholangiocarcinoma (bile duct cancer) has been observed in Thailand. This usually rare cancer has been associated with infection with the human liver fluke, Opisthorchis viverrini. Secretions of the parasite that interact with the host are thought to be a major component of its pathogenicity and proteolysis is a key biological activity of the secreted molecules. In this study, we present a molecular analysis of cysteine proteinase inhibitors (cystatins) of Opisthorchis viverrini. Six cDNA coding sequences of Opisthorchis viverrini cystatins, OvCys1-6, were cloned from the adult stage of the parasite using RT-PCR. Based on their sequences, OvCys1 and OvCys2 are classified as type 1 cystatins, while OvCys3-6 are classified as type 2 cystatins, with each containing a signal peptide and only one C-terminal disulfide bond. Their C-terminal region sequences are diverse compared with other cystatin members. Cystatins OvCys1, 3 and 4 were found in crude worm extracts and excretory-secretory (ES) products from the adult parasite using Western blot detection, while the other isoforms were not. Thus, OvCys1, 3 and 4 were selected for inhibition analysis and immune reactivity with Opisthorchis viverrini-infected hamster sera. OvCys1, 3, and 4 inhibited mammalian cathepsin L more effectively than cathepsin B. The pH range for their full activity was very wide (pH 3-9) and they were heat stable for at least 3 h. Unlike Fasciola gigantica cystatins, they showed no immune reactivity with infected hamster sera based on indirect ELISA. Our findings suggest that Opisthorchis viverrini cystatins are not major antigenic components in the ES product of this parasite and that other effects of Opisthorchis viverrini cystatins should be investigated.

Molecular Cloning and Characterization of a Fasciola gigantica Nuclear Receptor Subfamily 1 (FgNR1).

Fasciola gigantica, a giant liver fluke, causes tremendous loss to the livestock economy in several regions throughout the world. The situation of drug resistance has been emerging increasingly; therefore, novel drugs and drug targets need to be discovered. The adult F. gigantica inhabits the major bile ducts where bile salts accumulate-these are steroid-like molecules that mediate several physiological processes in organisms through interacting with their specific nuclear receptors. However, the molecular mechanism of the interaction in the parasitic organisms have not been clearly understood. In this study, putative nuclear receptor subfamily 1 of F.&amp;nbsp;gigantica (FgNR1) was identified. Nucleotide and amino acid sequences of the FgNR1 homolog were obtained from the transcriptome of F. gigantica and predicted for properties and functions using bioinformatics. The full-length cDNA was cloned and expressed in the bacterial expression system and then used for immunization. Western analysis and immunolocalization suggested that FgNR1 could be detected in the crude worm antigens and was highly expressed in the caeca and testes of the adult parasite. Moreover, the bile could significantly activate the expression of FgNR1 in cultured parasites. Our results indicated that FgNR1 has high potential for the development of a novel anthelminthic drug in the future.

Animal welfare and zoonosis risk: anti-Trichinella antibodies in breeding pigs farmed under controlled housing conditions

BackgroundDomesticated pigs are the main source of Trichinella sp. infections for humans, particularly when reared in backyards or free-ranging. In temperate areas of southern Europe, most pigs are farmed under controlled housing conditions, but sows and sometimes fattening pigs have access to outdoors to improve animal welfare. The aim of the present study was to investigate whether outdoor access of breeding pigs farmed under controlled housing conditions can represent a risk for Trichinella sp. transmission when the farm is located in an agricultural area interspersed with wooded areas and badlands, where Trichinella spp. could be present in wildlife.MethodsSerum samples were collected from 63 breeding sows and one boar before and after their access to an open fenced area for 2 months and from 84 pigs that never had outdoor access. Samples were screened for anti-Trichinella antibodies by ELISA, and positive sera were confirmed using Western blot (Wb) excretory/secretory antigens. To detect Trichinella sp. larvae, muscle tissues from serologically positive and negative pigs were tested by artificial digestion.ResultsThirteen (20.6%) sows and one boar tested positive with both ELISA and Wb. No larvae were detected in muscle samples of serologically positive and serologically negative pigs. Positive serum samples were then tested by Wb using crude worm extract as antigens. The Wb banding pattern displayed was that characteristic of encapsulated species (Trichinella spiralis or Trichinella britovi).ConclusionsThe detection of anti-Trichinella antibodies without larvae in the pig muscles, supported by epidemiological data, suggests that pigs may have been exposed to T. britovi. This study stresses the importance of instigating monitoring systems at farm level to prevent Trichinella sp. transmission and to investigate, through a landscape parasitological study, the suitability of a site before the planting of a high containment level pig farm in which the sows can have outside access to improve their welfare during pregnancy.Graphical abstract

The detection of anti-Trichinella antibodies in free-ranging Nebrodi Regional Park black pigs from Sicily, Italy, suggests the circulation of Trichinella britovi in the island.

• Zoonotic nematodes of the genus Trichinella are known to occur in countries bordering the Mediterranean Sea. For example, Trichinella spiralis was reported in pigs and humans at the turn of the Second World War in Sicily and Trichinella britovi in pigs, red foxes and dogs from Corsica and Sardinia since 2004. Following the discovery of T. britovi in Corsica and Sardinia, a question arose as to whether this species was also present in Sicily which is located only 3.14 km from continental Italy. To address this query, we investigated the presence of anti- Trichinella antibodies in the serum of Nebrodi black pigs, a breed that is bred in the wild in the Nebrodi Regional Park, a protected area of the island rich in flora and fauna. Blood samples were collected from 112 Nebrodi black pigs from five farms. Sera were tested by ELISA and ELISA positive sera were confirmed by Western blot (Wb) using excretory/secretory antigens. Eighteen (16.1%) serum samples belonging to 17 fattening pigs and 1 boar tested positive by Wb. Positive sera tested by Wb using crude worm extract antigens (CWE), displayed a banding pattern similar to the CWE-Wb pattern of T. spiralis and T. britovi reference pig sera but different to that of T. pseudospiralis reference pig sera. No larvae were detected in muscles of serologically positive pigs by artificial digestion. The presence of anti- Trichinella antibodies in the absence of larvae in the muscles, suggests that the pigs were infected with T. britovi and not T. spiralis whose larvae survive in the muscles for at least two years. These results suggest that T. britovi is circulating in Nebrodi Regional Park in Sicily. ELISA testing may constitute a suitable tool for large-scale screening of Trichinella spp. infection in free-ranging pigs, when ELISA-positive sera are confirmed by Wb. Free-ranging animals can act as sentinels for the presence of zoonotic nematodes of the genus Trichinella in wildlife. • Free-ranging pig sentinels for Trichinella spp. surveillance in wildlife. • Combined serological tests are informative for Trichinella spp. epidemiology. • Sicily is the third Mediterranean islands with Trichinella infection in wildlife.

Dynamics of serological responses to defined recombinant proteins during Schistosoma mansoni infection in mice before and after the treatment with praziquantel.

To eliminate schistosomiasis, appropriate diagnostic tests are required to monitor its prevalence and transmission, especially in the settings with low endemicity resulting from the consecutive mass drug administration. Antibodies that react with either crude soluble schistosome egg antigens or soluble worm antigen preparations have been used to monitor infection in low-prevalence regions. However, these detection methods cannot discriminate current and past infections and are cross-reactive with other parasites because both antigens contain numerous proteins and glycans from schistosomes, and standard preparations need maintenance of the life cycle of the schistosome. To evaluate the potential utility of nine recombinant Schistosoma mansoni proteins as single defined antigens for serological diagnosis, we monitored the kinetics of antibodies to each antigen during S. mansoni infection in mice before and after the treatment with praziquantel. C57BL/6 mice were infected with 50 cercariae. The levels of immunoglobulin G (IgG) raised against five recombinant antigens (RP26, sm31, sm32, GST, and LAP1) significantly increased as early as 2–4 weeks after infection and rapidly declined by 2 weeks after the treatment, whereas those raised against crude S. mansoni egg antigens or other antigens remained elevated long after the treatment. The IgG1 raised against RP26, sm31, and serpin decreased after the treatment with praziquantel, whereas the IgE raised against serpin declined strikingly after the treatment. This study clarifies the dynamics of the serological responses to recombinant S. mansoni proteins during infection and after the treatment with praziquantel and identifies several candidate antigens with potential utility in the monitoring and surveillance of schistosomiasis toward the elimination of schistosomiasis.

Antigen Recognition Patterns of Intestinal Capillariasis Using Immunoblot-Based Serodiagnosis.

Diagnosis of intestinal capillariasis depending on microscopic detection of parasitic stages is of low sensitivity, especially in cases with low worm burden. There is a necessity to develop sensitive and specific diagnostic tools of capillariasis for early treatment to avoid complications. Western blot (WB) technique showed promising results for antigen recognition patterns in several parasitic infections. This study is directed to identify and evaluate relevant proteins of intestinal capillariasis crude worm antigens using WB immunodiagnosis. Capillaria crude worm antigens were extracted and analyzed using SDS-PAGE. Sixty serum samples belonging to 3 groups (20 individuals each) were included; Group I (shedding Capillaria in feces), Group II (infected with other parasites) and Group III (negative parasitological results). Reactivity of the resulting bands of Capillaria with serum samples was analyzed using WB technique. Thirty-two immunoreactive bands were detected in WB analysis representing recognition of proteins with molecular weights (MW) varying from 19 to 110kDa. Immunodominant proteins of 23.5, 31, 36.5, 40.5 and 44kDa were recognized, respectively, in 35%, 30%, 85%, 95% and 75% of sera from patients with confirmed capillariasis and in 30%, 25%, 35%, 25%, and 20% of sera from those infected with other parasitic infections. One serum sample from group III gave reaction with 31kDa band. Immunodiagnosis of intestinal capillariasis using WB proved that 23.5, 31, 36.5, 40.5 and 44kDa bands could be considered useful tools for diagnosis of capillariasis.

A novel cystatin derived from Trichinella spiralis suppresses macrophage-mediated inflammatory responses

Trichinella spiralis can modulate host immune responses to retain a suitable environment for its long-term survival. Incidentally, the parasite elicits regulatory effects through immunomodulatory molecule release, which can suppress host inflammation and may be used for the treatment of unrelated inflammatory diseases in someday. Here we identified and characterized a novel T. spiralis cystatin (TsCstN), which inhibits inflammation mediated by LPS-treated macrophages.Proteins contained in the excretory–secretory (ES) product of muscle-stage T. spiralis (ES-L1) were fractionated, and each was treated with mouse bone marrow-derived macrophages (mBMDMs) before LPS stimulation. The fractions that exhibited high immunomodulatory property by decreasing pro-inflammatory cytokines or increasing anti-inflammatory cytokines were identified by mass spectrometry. Incidentally, the conserved hypothetical protein (Tsp_04814) was selected for further characterization as it presented the most significant MS score. An annotation of Tsp_04814 using protein structural homology comparison suggested that it has high structural similarity to human cystatin E/M (TM score 0.690). The recombinant T. spiralis novel cystatin (rTsCstN) was expressed in Escherichia coli at a molecular weight of approximately 13 kDa. Mouse anti-rTsCstN polyclonal antibody (pAb) could detect native TsCstN in crude worm antigens (CWA) and ES-L1 and be predominantly localized in the stichosome and subcuticular cells. rTsCstN inhibited cysteine proteases in vitro, especially cathepsin L, at an optimal pH of 6. Besides, rTsCstN could be internalized into mBMDMs, which were mostly distributed in the cytoplasm and lysosome both before and after LPS stimulation. To evaluate the rTsCstN immunomodulatory properties on mBMDMs, rTsCstN was incubated with mBMDM before LPS stimulation; this demonstrated that rTsCstN suppressed pro-inflammatory cytokine production and MHC class II expression.T. spiralis L1-derived TsCstN was characterized as a novel cysteine protease inhibitor. The protein elicits an anti-inflammatory property by suppressing pro-inflammatory cytokines and interfering with the antigen presentation process through depletion of MHC class II expression.

Molecular cloning and characterization of serine protease inhibitor from food-borne nematode, Gnathostoma spinigerum

Gnathostoma spinigerum is a causative agent of human gnathostomiasis and infects people residing in endemic areas as well as travelers. Cutaneous and visceral larval migrants cause clinical manifestations, resulting in severe morbidity and mortality. To survive in hosts, these parasites have evolved various immune evasion mechanisms, including the release of regulatory molecules. Serine protease inhibitors (serpins) that are present in many parasitic helminths are proteins suspected of suppressing host serine protease-related digestion and immune responses. In this study, the serpin secreted by G. spinigerum (GsSerp) was characterized using bioinformatics and molecular biology techniques. The bioinformatics revealed that GsSerp contains 9 helices, 3 β-sheets, and a reactive central loop, which are conserved structures of the serpin superfamily. Recombinant GsSerp (rGsSerp) was expressed in E. coli (molecular weight, 39 kDa) and could inhibit chymotrypsin. Mouse polyclonal antibody against GsSerp could detect the native GsSerp in crude worm antigen but not the excretory–secretory product (ES) of infective-stage larva (aL3Gs). Moreover, the expression of GsSerp in the aL3Gs tissue was located in the hemolymph and intestinal tissue, indicating its role in parasite homeostasis. Our findings may help develop effective strategies for preventing and controlling gnathostomiasis.

Molecular characterization and functional analysis of the Schistosoma mekongi Ca2+-dependent cysteine protease (calpain)

BackgroundSchistosoma mekongi, which causes schistosomiasis in humans, is an important public health issue in Southeast Asia. Treatment with praziquantel is the primary method of control but emergence of praziquantel resistance requires the development of alternative drugs and vaccines. Calcium-dependent cysteine protease (calpain) is a novel vaccine candidate that has been studied in S. mansoni, S. japonicum, and protozoans including malaria, leishmania and trypanosomes. However, limited information is available on the properties and functions of calpain in other Schistosoma spp., including S. mekongi. In this study, we functionally characterized calpain 1 of S. mekongi (SmeCalp1).ResultsCalpain 1 of S. mekongi was obtained from transcriptomic analysis of S. mekongi; it had the highest expression level of all isoforms tested and was predominantly expressed in the adult male. SmeCalp1 cDNA is 2274 bp long and encodes 758 amino acids, with 85% to 90% homology with calpains in other Schistosoma species. Recombinant SmeCalp1 (rSmeCalp1), with a molecular weight of approximately 86.7 kDa, was expressed in bacteria and stimulated a marked antibody response in mice. Native SmeCalp1 was detected in crude worm extract and excretory-secretory product, and it was mainly localized in the tegument of the adult male; less signal was detected in the adult female worm. Thus, SmeCalp1 may play a role in surface membrane synthesis or host–parasite interaction. We assessed the protease activity of rSmeCalp1 and demonstrated that rSmeCalp1 could cleave the calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin, that was inhibited by calpain inhibitors (MDL28170 and E64c). Additionally, rSmeCalp1 could degrade the biological substrates fibronectin (blood clotting protein) and human complement C3, indicating important roles in the intravascular system and in host immune evasion.ConclusionsSmeCalp1 is expressed on the tegumental surface of the parasite and can cleave host defense molecules; thus, it might participate in growth, development and survival during the entire life-cycle of S. mekongi. Information on the properties and functions of SmeCalp1 reported herein will be advantageous in the development of effective drugs and vaccines against S. mekongi and other schistosomes.

Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Kit and In-House Fasciola gigantica Cysteine Proteinases-Based Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Fascioliasis.

Fascioliasis, caused by Fasciola hepatica and Fasciola gigantica infection, is a major food-borne trematodiasis in many places of the world, with the central region of Vietnam being reported as a highly endemic area. Stool examination for Fasciola eggs is not a sensitive method, and immunodiagnostic methods are preferable. We investigated various enzyme-linked immunosorbent assays (ELISAs) to evaluate their efficacy for fascioliasis diagnosis. Test sera used are primarily screened using an ELISA kit produced in Vietnam (VN kit; Viet Sinh Chemical Producing & Trading Co. Ltd., Ho Chi Minh City, Vietnam): Seropositive individuals having symptoms compatible with fascioliasis were regarded as clinically diagnosed fascioliasis cases. A commercial Fasciola IgG ELISA kit from Diagnostic Automation/Cortez Diagnostics, Inc. (USA kit; Woodland Hills, CA), which has been commonly used in Vietnam, was assessed and compared with in-house ELISA systems, including a cystatin-capture (CC) ELISA using crude worm extract (CWE) and an indirect ELISA using a synthetic peptide Ac-TPTCHWECQVGYNKTYDEE-NHMe designed from the F. gigantica cathepsin B (FgCB5) molecule. The USA kit was suitable for routine diagnosis after recalibration of the manufacturer's suggested cutoff point. Cystatin-capture ELISA with CWE provided good sensitivity and specificity with perfect agreement to the results of the USA kit. In dot-blot ELISA, recombinant FgCB5 reacted more strongly with human antisera than did other F. gigantica antigens tested. Enzyme-linked immunosorbent assay using the synthetic peptide fragment of the FgCB5 exhibited nearly 80% sensitivity and specificity, but the test results showed low agreement with CC-ELISA or the USA kit. In conclusion, the commercially available Fasciola IgG ELISA kit from the United States and the in-house CC ELISA using CWE are suitable for practical diagnosis for fascioliasis.

Differentiation of Trichinella species (Trichinella spiralis/Trichinella britovi versus Trichinella pseudospiralis) using western blot

BackgroundTrichinellosis is a meat-borne zoonotic disease caused by parasites of the genus Trichinella. To date, 12 taxa have been described. The identification of Trichinella species is crucial in order to identify the possible source of infection, the geographical origin of the parasite and to assess risk of infection for domestic pigs and humans. Specific identification of the etiological agent is not always feasible using direct methods since the source of infection can be untraceable. The aim of this study was to develop a diagnostic tool to infer the causative Trichinella species using western blot patterns of sera derived from infected animal and human hosts.MethodsSera from mice experimentally infected with Trichinella spiralis, Trichinella britovi, Trichinella pseudospiralis and Trichinella papuae were tested by western blot using homologous and heterologous crude worm extracts (CWE) and a highly sensitive detection system based on chemiluminescence. In addition, sera from pigs experimentally infected with T. spiralis, T. britovi and T. pseudospiralis and from patients with confirmed T. spiralis, T. britovi and T. pseudospiralis infections, were also included.ResultsSera from mice infected with one Trichinella species reacted with CWE proteins from all four investigated species. Likewise, sera derived from pigs and humans infected with one Trichinella species reacted with CWE proteins from all the three investigated species. Using T. spiralis CWE, sera from T. pseudospiralis-infected hosts yielded a characteristic pattern of reactivity using Wb, which differed to that produced by T. spiralis/T. britovi- or T. papuae-infected host sera.ConclusionsThe present study suggests that western blot using T. spiralis CWE may be a useful tool to distinguish Trichinella infections caused by T. pseudospiralis from those caused by T. spiralis or T. britovi. This method may support epidemiological investigations, particularly when the source of infection is not traceable.

Onchocerca volvulus infection and serological prevalence, ocular onchocerciasis and parasite transmission in northern and central Togo after decades of Simulium damnosum s.l. vector control and mass drug administration of ivermectin.

BackgroundMass drug administration (MDA) of ivermectin has become the main intervention to control onchocerciasis or “river blindness”. In Togo, after many years of MDA, Onchocerca volvulus infection has declined dramatically, and elimination appears achievable, but in certain river basins the current situation remains unknown. We have conducted parasitological, serological, ophthalmological, and entomological assessments in northern and central Togo within the river basins of Ôti, Kéran and Mô.Methodology/Principal findingsExaminations were completed in 1,455 participants from 11 onchocerciasis sentinel villages, and O. volvulus transmission by Simulium damnosum sensu lato (s.l.) was evaluated. In children (aged 1–10 years), the prevalence of microfilariae (Mf) was 2.3% and in adults it ranged from 5.1 to 13.3%. Positive IgG4 responses to O. volvulus adult (crude) worm antigen (OvAg) and the recombinant Ov16 antigen were in all-ages 48.7% and 34.4%, and 29.1% and 14.9% in children, respectively. In the river basin villages of Kéran, Mô and Ôti, the IgG4 seroprevalences to OvAg in children were 51.7%, 23.5% and 12.7%, respectively, and to the Ov16 antigen 33.3% (Kéran) and 5.2% (Ôti). Onchocerciasis ocular lesions (punctate keratitis, evolving iridocyclitis and chorioretinitis) were observed in children and young adults. O. volvulus-specific DNA (Ov150) was detected by poolscreen in vector samples collected from Tchitchira/Kéran(22.8%), Bouzalo/Mô(11.3%), Baghan/Mô(2.9%) and Pancerys/Ôti(4.9%); prevalences of O. volvulus infection in S. damnosum s.l. were, respectively, 1%, 0.5%, 0.1% and 0.2%.Conclusions/SignificanceIn the northern and central river basins in Togo, interruption of O. volvulus transmission has not yet been attained. Patent O. volvulus infections, positive antibody responses, progressive ocular onchocerciasis were diagnosed, and parasite transmission by S. damnosum s.l. occurred close to the survey locations. Future interventions may require approaches selectively targeted to non-complying endemic populations, to the seasonality of parasite transmission and national onchocerciasis control programs should harmonize cross-border MDA as a coordinated intervention.

Young mice expel the tapeworm Hymenolepis diminuta and are protected from colitis by triggering a memory response with worm antigen.

Infection with helminth parasites reduces the severity of concomitant inflammatory disease in adult mice. There is an alarming increase of inflammatory bowel disease (IBD) in children. It is important to determine whether helminth therapy would be of value in pediatric IBD and whether triggering immunological memory to the worm would be anticolitic. Three-week-old (young) and eight-week-old (adult) Balb/c mice were infected with H. diminuta, and infectivity and T helper 2 (Th2) immunity were assessed. Other mice received H. diminuta with or without a crude worm extract ( HdE) 28-42 days postinfection (dpi) with or without dinitrobenzene sulphonic acid [DNBS, 1.5 mg (young) or 3 mg (adults), ir], and colitis was assessed 72 h later. Infected young mice developed Th2 immunity and expelled H. diminuta; expulsion was delayed by ~2 days compared with adult mice. Colitis, as gauged by macroscopic disease and histopathology scores, was less severe in young mice infected 10 days, but not 8 days, before DNBS. Protection against DNBS-induced colitis was accompanied by an increased capacity to make interleukin (IL)-4 and IL-10. Mice infected with H. diminuta were not protected from DNBS-colitis when challenged 28 days later; however, injection of these mice with HdE coincident with DNBS resulted in less disease and increased splenic IL-4 and IL-10. Using a boost (500 μg HdE, 28 dpi) and repeat HdE (100 μg, 42 dpi) regimen with infected mice suppressed DNBS-colitis, as did adoptive transfer of splenic CD4+ T cells from infected mice with low-dose HdE challenge. Should these data translate to IBD, then helminth therapy could be of value in pediatric-onset IBD, and defining the antigen(s) that elicit antihelminth immunological memory could serve as an anticolitic approach in previously infected individuals. NEW & NOTEWORTHY This study demonstrates that juvenile mice are protected from colitis by infection with the tapeworm Hymenolepis diminuta and that using worm antigen to trigger an immunological memory response in previously infected mice can be used to limit the severity of colitis.

Evaluation of Schistosoma japonicum thioredoxin peroxidase-1 as a potential circulating antigen target for the diagnosis of Asian schistosomiasis.

Asian schistosomiasis caused by Schistosoma japonicum is a serious zoonotic disease endemic in China, the Philippines and parts of Indonesia. Mass drug administration in endemic areas resulted to decline in disease severity and intensity. The low intensity of infection limits the use of current parasitological methods for schistosomiasis diagnosis. Detection of parasite circulating antigens might provide more informative result as it may indicate the true status of infection. In this study, S. japonicum thioredoxin peroxidase-1 (SjTPx-1) a 22 kDa secreted antioxidant enzyme expressed throughout the life stages of the parasite was evaluated for its potential use as a biomarker for schistosomiasis japonica infection. Rabbit polyclonal antibody and mouse monoclonal antibodies (mAbs) were raised against the recombinant SjTPx-1 (rSjTPx-1). The antibodies produced against the recombinant antigen was confirmed to detect the native SjTPx-1 in crude adult worm lysate. Likewise, the specific binding of mAbs to parasite TPx-1 and not to mammalian peroxiredoxin-1 orthologues was also confirmed. The double antibody sandwich ELISA developed in this study was able to detect at least 1 ng/ml of rSjTPx-1. In addition, this method was able to detect the antigen from all serum samples of experimentally infected rabbit and mice. The diagnostic potential of SjTPx-1 in human clinical samples was also evaluated, in which 4 out of 10 stool-confirmed serum samples had detectable levels of the antigen. The results suggest that SjTPx-1 can be a potential biomarker for Asian zoonotic schistosomiasis.

THE ANTIGENIC RELATIONSHIP BETWEEN SCHISTOSOMA MANSONI AND ITS INTERMEDIATE SNAIL HOST.

Schistosomiasis is a public health problem, in many developing: countries including Egypt, Determination of the antigenic relationship between S mansoni and its intermediate snail host (IMH) Biomphalaria alexandrina can open a new field for diagnosis and control of the dis- ease. In the present study infected and non-infected B. alexandrina foot and visceral hump tissue as well as S. mansion crude Ag (SWAg) were fractionated using SDS-PAGE. It's specific and cross reacted protein fractions were determine using EITB versus experimentally prepared mice hyper immune sera (HIS) versus each antigen. After treatment of fractionated S.mansoni crude worm antigens (SWAg) versus HIS produced after vaccination of mice by the same Ag, 8 kda protein fractions ranged from 35-140 kda were reacted specifically. Treatment of fractionated B.alexandrina infected and non-infected foot and visceral hump Ag versus previous HIS revealed presence of common polypeptides bands between SWAg and non-infected snail antigens. The fraction at 135 kda, 68 kda, were detected in all cases, while that at 40-42 kda and that at 35 kda was diagnosed in SWAg and that of infected snails only. The fraction at 68 kda was reacted specifically between SWAg and all tested fractionated snail antigens either that of foot or visceral hump when they treated separately by HIS of mice vaccinated by each snail A eparately. The fraction at 135 kda was common between SWAg and snail (infected and non-infected) visceral hump antigen. The fraction at the level of 110 kda was diagnosed inSWAg, in non-infected foot antigen and visceral hump Ag. The fraction at the level of 46-48 (da are common between SWAg and snail foot and visceral hump Ag after treated by HIS of mice vaccinated by foot Ag, Presence of common antigenic fractions between snail tissues and Schistosoma species can prefer an easily source of antigen valuable for diaguosis or vaccination as well as can be considered as new tool for determination to the snail IMH of new discovered trematode Darasites.

Molecular and immunological characterization of cathepsin L-like cysteine protease of Paragonimus pseudoheterotremus.

Cathepsin L is a cysteine protease belonging to the papain family. In parasitic trematodes, cathepsin L plays essential roles in parasite survival and host-parasite interactions. In this study, cathepsin L of the lung fluke Paragonimus pseudoheterotremus (PpsCatL) was identified and its molecular biological and immunological features characterized. A sequence analysis of PpsCatL showed that the gene encodes a 325-amino-acid protein that is most similar to P. westermani cathepsin L. The in silico three-dimensional structure suggests that PpsCatL is a pro-enzyme that becomes active when the propeptide is cleaved. A recombinant pro-PpsCatL lacking the signal peptide (rPpsCatL), with a molecular weight of 35kDa, was expressed in E. coli and reacted with P. pseudoheterotremus-infected rat sera. The native protein was detected in crude worm antigens and excretory-secretory products and was localized in the cecum and in the lamellae along the intestinal tract of the adult parasite. Enzymatic activity of rPpsCatL showed that the protein could cleave the fluorogenic substrate Z-Phe-Arg-AMC after autocatalysis but was inhibited with E64. The immunodiagnostic potential of the recombinant protein was evaluated with an enzyme-linked immunosorbent assay (ELISA) and suggested that rPpsCatL can detect paragonimiasis with high sensitivity and specificity (100 and 95.6%, respectively). This supports the further development of an rPpsCatL-ELISA as an immunodiagnostic tool.

Immunodiagnosis of Haemonchus contortus infection in sheep by counter immuno-electrophoresis using somatic antigen.

The present study was undertaken for the immuno-diagnosis of Haemonchus contortus infection in sheep by counter immuno-electrophoresis (CIEP) using crude somatic antigen. Adult worms of H. contortus were found in 57 out of 100 abomasums screened. A total of 250 serum samples which includes, 100 serum from local abattoir in and around Shimoga region from the sheep from which the abomasums were collected for H. contortus worms and 150 serum from the migratory sheep were collected to detect the circulating antibody of H. contortus by CIEP using crude whole worm somatic antigen. Out of 100 abomasums screened, 18 were heavily infected with adult worms of H. contortus and their serum samples showed positive reaction with clear thick precipitin line with crude somatic antigen of H. contortus by CIEP and 39 samples showed light precipitin line, which were moderately infected with adult worms of H. contortus during necropsy. However, the 12 serum samples showed positive reaction with clear precipitin line, but their abomasums did not show H. contortus and the remaining 31 were not harbouring any adult worms of H. contortus and no precipitin line observed by CIEP. The sensitivity and specificity of CIEP was found 100 and 72.09%, respectively. In the present study, 62% sero-prevalence was observed with CIEP among 150 serum samples from migratory sheep screened for antibodies of H. contortus using crude whole worm somatic antigen of H. contortus.

Similarity of a 16.5 kDa tegumental protein of the human liver fluke Opisthorchis viverrini to nematode cytoplasmic motility protein

Opisthorchis viverrini is the causative agent of human opisthorchiasis in Thailand and long lasting infection with the parasite has been correlated with the development of cholangiocarcinoma. In this work we have molecularly characterized the first member of a protein family carrying two DM9 repeats in this parasite (OvDM9-1). InterPro and other protein family databases describe the DM9 repeat as a protein domain of unknown function that has been first noted in Drosophila melanogaster. Two paralogous proteins have been partially characterized in the genus Fasciola, Fasciola hepatica TP16.5, a novel tegumental antigen in human fascioliasis and, recently F. gigantica DM9-1, a parenchymal protein with structural similarity to nematode cytoplasmic motility protein (MFP2). In this study, we show further evidence that this family of trematode proteins is related to MFP2 in sequence and structure. Soluble recombinant OvDM9-1 was used for structural analyses and for production of specific antisera. The native protein was detected in soluble and insoluble crude worm extracts and in seemingly various oligomeric forms in the latter. The potential for oligomerization was supported by cross-linking experiments of recombinant OvDM9-1. Structure prediction suggested a β-rich secondary structure of the protein and this was supported by a circular dichroism analysis. Molecular modeling in Phyre2 identified both MFP2 domains as distant homologs of OvDM9-1. The protein was located in tegumental type tissue and the cecal epithelium in the mature parasite. Recombinant OvDM9-1 was used as target in indirect ELISA but sera from infected hamsters showed only marginal reactivity towards it. It is proposed that OvDM9-1 and other members of this protein family have a role in cellular transport through functions on the cytoskeleton.

Fasciola gigantica cathepsin B5 is an acidic endo- and exopeptidase of the immature and mature parasite.

Cysteine proteases of the liver fluke Fasciola have been described as essential molecules in the infection process of the mammalian host. Destinct cathepsin Bs, which are already expressed in the metacercarial stage and released by the newly excysted juvenile are major actors in this process. Following infection their expression is stopped and the proteins will not be detectable any longer after the first month of development. On the contrary, the novel cathepsin B5 of Fasciola gigantica (FgCB5) described in this work was also found expressed in later juvenile stages and the mature worm. Like all previously described Fasciola family members it was located in the cecal epithelium of the parasite. Western blot analysis of adult antigen preparations detected procathepsin B5 in crude worm extract and in small amounts in the ES product. In support of these data, the sera of infected rabbits and mice were reactive with recombinant FgCB5 in Western blot and ELISA. Biochemical analysis of yeast-expressed FgCB5 revealed that it has properties of a lysosomal hydrolase optimized for activity at acid pH and that it is able to efficiently digest a broad spectrum of host proteins. Unlike previously characterized Fasciola family members FgCB5 carries a histidine doublet in the occluding loop equivalent to residues His110 and His111 of human mature cathepsin B and consequently showed substantial carboxydipeptidyl activity which depends on these two residues.