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Background: Several Tritrichomonas species have been found in mammalian hosts. Among these trichomonads T. foetus is often found in the urogenital tract of cattle and the gastrointestinal tract of the domestic cat, resulting in sexually transmitted bovine trichomonosis and fecal-orally transmitted feline trichomonosis, respectively. The aims of the current study were to molecularly characterize clinical isolates of T. foetus in cattle populations in Wyoming, South Dakota, and Montana of the United States of America and to phylogenetically analyze Tritrichomonas species of mammalian hosts. Results: DNA sequencing of rRNA genes showed over 99% identity of the newly described isolates to other bovine isolates. Further, T. foetus isolates of various mammalian hosts originated in different geographic regions worldwide were clustered into two well-defined clades by phylogenetic analysis of rRNA and cysteine protease 2 genes. Clade I consisted of isolates originated from cattle, pig, and human whereas clade II contained isolates of cat and dog. Conclusion: It is concluded that all mammalian Tritrichomonas spp. apparently belong to T. foetus. Analysis of more sequences is warranted to support this conclusion. [ABSTRACT FROM AUTHOR]

Our previous studies demonstrated that total polysaccharides isolated from Atractylodis macrocephalae Koidz. ( RAMPtp ) had a therapeutic effect on bovine subclinical mastitis and an immunomodulatory effect on the lymphocytes from the supramammary lymph node (SMLN) in cows. To investigate the potential molecules involved in the immunomodulation, the present study identified 243 differentially expressed genes (DEGs) in RAMPtp- stimulated SMLN lymphocytes by RNA-seq. GO/KEGG enrichment analyses identified 47 significantly enriched gene ontology (GO) terms and 9 canonical pathways. These findings indicated that RAMPtp might stimulate lymphocytes to modulate productions of cytokines and chemokines through multiple compounds, targets and pathways. In particular, the Jak-STAT signaling pathway might be primarily involved in the immunomodulation. The therapeutic effect of RAMPtp on bovine subclinical mastitis may be attributed to activation of SMLN lymphocytes by injection of the polysaccharides in the area of SMLN in cows. [ABSTRACT FROM AUTHOR]

Abstract: The nematode Haemonchus contortus (H. contortus) is one of the most pathogenic and economically important parasites of sheep. A 24kDa protein is one of the important components in H. contortus excretory/secretory (ES), which was shown to have important biological function. In our research, the cDNA of its open reading frame (ES24) was obtained and analyzed. Then the ES24 was sub-cloned into pET-30a expression vector. The recombinant vector that codes hexahistidyl peptide fusion protein (His-ES24) was transformed into Escherichia coli BL21 (DE3) strain. After induction, a high expression level of His-ES24 was found at 6h taking about 26% of the total bacterial protein analyzed by gel thin-layer scanning. The expressed His-ES24 was purified and then used in an enzyme-linked immunosorbent assay (ELISA) to detect specific antibodies in serum samples. The ELISA was able to differentiate between H. contortus-infected sheep serum and Fasciola hepatica-infected sheep serum or non-infected sheep serum. No cross-reaction was observed in sheep sera that have been experimentally infected with F. hepatica. A total of 153 field sheep serum samples conserved in our laboratory were examined using the His-ES24 ELISA, and 82 (53.6%) of them were found seropositive to H. contortus. Our results demonstrate that the prokaryotic-expressed His-ES24 might be a useful diagnostic reagent for epidemiological studies of H. contortus in sheep. [Copyright &y& Elsevier]

Abstract: Three-day-old specific-pathogen-free chickens were subcutaneously immunized with Eimeria tenella recombinant 5401 antigen (100μg per chicken) with (0.25, 0.5 or 1.0mg per dose) or without ginsenosides, and boosted with the same dosage 14 days later. The chickens were challenged with 6×104 homologous sporulated oocysts 14 day after the booster. The specific antibody response and lymphocyte proliferation in response to Con A were measured before and 7, 14, 21, 28, 35, 42 days after the immunization. Oocyst output, mortality, and lesion scores were measured to evaluate the protective effects of the immunization. The vaccine containing 0.5 or 1.0mg ginsenosides per dose induces higher antibody response and lymphocyte proliferation in response to Con A than the vaccine without ginsenosides or containing 0.25mg per dose. The oocyst output indicated that recombinant 5401 antigen with ginsenosides (0.5 and 1.0mg per dose) gave a protection rate of 59.38 and 62.5%, respectively. The lesion score in the group vaccinated with recombinant 5401 antigen with 0.5 or 1.0mg ginsenosides per dose were significantly lower than in group without ginsenosides or containing 0.25mg per dose. Therefore, we conclude that ginsenosides have strong adjuvant effects at a dose of 0.5 or 1.0mg when mixed with E. tenella recombinant 5401 antigen, and has a potential as an adjuvant in chicken vaccine. [Copyright &y& Elsevier]

Abstract: The efficacy of an oral DNA vaccine carrying the Eimeria tenella 5401 antigen gene delivered by attenuated Salmonella typhimurium was examined in an experimental challenge study. The DNA vaccine preparation was made by transforming the recombinant plasmid pcDNA3-5401 into the attenuated S. typhimurium strain (Dam− and PhoP−) (designated hereafter as ZJ111/pcDNA3-5401). The chickens were randomly divided into six groups, 50 per group. Group A were given PBS as control. Chickens in group B were fed with 108 colony forming units (CFU) of attenuated S. typhimurium carrying pcDNA3. Group C were immunised with 100μg of the recombinant 5401 protein via intramuscular injection. Groups D to F orally received ZJ111/pcDNA3-5401 at doses of 107, 108 and 109 CFU per chicken, respectively. All immunisations were boosted 2 weeks later. The immunised chickens were challenged with 6×104 homologous sporulated oocysts 14 days after the second immunisation. No significant differences in body weight were detected between the groups before immunisation and at week 4 after the booster immunisation. The ZJ111/pcDNA3-5401 was eventually eliminated from the spleen and liver on week 6 post-immunisation. The plasmid pcDNA3-5401 was stably maintained in over 80% of the attenuated S. typhimurium population after 100 generations of growth in antibiotic-free media. Oral immunisation of chickens with ZJ111/pcDNA3-5401 elicited specific humoral responses and stimulated proliferation of peripheral blood lymphocytes. The lymphocyte proliferation response was significantly higher in all vaccinated groups than in the control chickens. Antibody response was significantly lower in group C than in groups immunised with strain ZJ111/pcDNA3-5401. Vaccination with the strain ZJ111/pcDNA3-5401 at 108 (group E) and 109 (group F) CFU per chicken provided 55.0 and 57.5% protection against E. tenella challenge, respectively. These results have important implications for the development of DNA vaccines against avian coccidiosis by bacteria-vectored oral delivery system. [Copyright &y& Elsevier]

Background: Human toxocariasis is a neglected parasitic disease characterised by the syndromes visceral, cerebral, and ocular larva migrans. This disease is caused by the migrating larvae of Toxocara roundworms from dogs and cats, affecting 1.4 billion people globally. Via extracellular vesicles (EVs), microRNAs have been demonstrated to play roles in host–parasite interactions and proposed as circulating biomarkers for the diagnosis and follow-up of parasitic diseases. Methods: Small RNA-seq was conducted to identify miRNAs in the infective larvae of T. canis and plasma EV-containing preparations of infected BALB/c mice. Differential expression analysis and target prediction were performed to indicate miRNAs involved in host–parasite interactions and miRNAs associated with visceral and/or cerebral larva migrans in the infected mice. Quantitative real-time polymerase chain reaction (PCR) was used to amplify circulating miRNAs from the infected mice. Results: This study reports host and parasite miRNAs in the plasma of BALB/c mice with visceral and cerebral larva migrans and demonstrates the alterations of these miRNAs during the migration of larvae from the livers through the lungs and to the brains of infected mice. After filtering unspecific changes in an irrelevant control, T. canis-derived miRNAs and T. canis infection-induced differential miRNAs are predicted to modulate genes consistently involved in mitogen-activated protein kinase (MAPK) signalling and pathways regulating axon guidance and pluripotency of stem in the infected mice with visceral and cerebral larva migrans. For these plasma circulating miRNAs predicted to be involved in host-parasite crosstalk, two murine miRNAs (miR-26b-5p and miR-122-5p) are experimentally verified to be responsive to larva migrans and represent circulating biomarker candidates for visceral and cerebral toxocariasis in BALB/c mice. Conclusions: Our findings provide novel insights into the crosstalk of T. canis and the mammalian host via plasma circulating miRNAs, and prime agents and indicators for visceral and cerebral larva migrans. A deep understanding of these aspects will underpin the diagnosis and control of toxocariasis in humans and animals. [ABSTRACT FROM AUTHOR]

Mine tailings have shown viability as the fine–grained layer in a capillary barrier structure for controlling acid mine drainage in a circular economy. Their saturated hydraulic conductivities (ksat) under wetting–drying cycles and freeze–thaw cycles remain unexplored. In this study, modified tailings with a weight ratio of 95:5 (tailings/hydrodesulfurization (HDS) clay from waste–water treatment) and an initial water content of 12% were used. The ksat of specimens was measured after up to 15 wetting–drying cycles, each lasting 24 h, with a drying temperature of 105 °C. The ksat for wetting–drying cycles decreased from 3.9 × 10−6 m/s to 9.5 × 10−7 m/s in the first three cycles and then stabilized in the subsequent wetting–drying cycles (i.e., 5.7 × 10−7 m/s–6.3 × 10−7 m/s). Increased fine particles due to particle breakage are the primary mechanism for the ksat trend. In addition, the migration of fines and their preferential deposition near the pore throat area may also promote this decreasing trend through the shrinking and potentially clogging–up of pore throats. This could be explained by the movement of the meniscus, increased salinity, and, subsequently, the shrinkage of the electrical diffuse layer during the drying cycle. Similar specimens were tested to measure ksat under up to 15 freeze–thaw cycles with temperatures circling between −20 °C and 20 °C at 12 h intervals. Compared to the untreated specimen (i.e., 3.8 × 10−6 m/s), the ksat after three freeze–thaw cycles decreased by 77.6% (i.e., 8.5 × 10−7 m/s) and then remained almost unchanged (i.e., 5.6 × 10−7 m/s–8.9 × 10−7 m/s) in subsequent freeze–thaw cycles. The increased fine grain content (i.e., 3.1%) can be used to explain the decreased ksat trend. Moreover, the migration of fines toward the pore throat area, driven by the advancing and receding of ice lens fronts and subsequent deposition at the pore throat, may also contribute to this trend. [ABSTRACT FROM AUTHOR]

Background: Components of excretory/secretory products (ESPs) of helminths have been proposed as vaccine targets and shown to play a role in modulating host immune responses for decades. Such research interest is further increased by the discovery of extracellular vesicles (EVs) in the ESPs of parasitic worms. Although efforts have been made to reveal the cargos of EVs, little is known about the proteomic differences between EVs and canonical ESPs released by parasitic worms from animals. Methods: The total ESPs of Haemonchuscontortus (barber's pole worm) were obtained by short-term in vitro culturing of young adult worms, and small EVs were isolated from ESPs using an ultracentrifugation method. Data-dependent acquisition (DDA) label-free Nano-LC–MS/MS was used to quantify the proteomic difference between small EVs and EV-depleted ESPs of H.contortus. Functional annotation and enrichment of the differential proteins were performed regarding cellular components, molecular functions, pathways, and/or biological processes. Results: A total of 1697 proteins were identified in small EVs and EV-depleted ESPs of H.contortus adult worms, with 706 unique proteins detected in the former and 597 unique proteins in the latter. It was revealed that proteins in small EVs are dominantly cytoplasmic, whereas proteins in EV-depleted ESPs are mainly extracellular; canonical ESPs such as proteases and small GTPases were abundantly detected in small EVs, and SCP/TAP-, DUF-, and GLOBIN domain-containing proteins were mainly found in EV-depleted ESPs. Compared with well-characterised proteins in small EVs, about 50% of the proteins detected in EV-depleted ESPs were poorly characterised. Conclusions: There are remarkable differences between small EVs and EV-depleted ESPs of H.contortus in terms of protein composition. Immune modulatory effects caused by nematode ESPs are possibly contributed mainly by the proteins in small EVs. [ABSTRACT FROM AUTHOR]

Background: Nematodes have lost the ability to synthesise necessary lipids de novo and have complementally evolved the capacity to acquire fatty acids and their derivatives from a diet or host animal. Nematode-specific fatty acid- and retinol-binding protein (FAR) family is one approach that facilitates lipid acquisition, representing an Achilles heel and potential target against roundworms of socioeconomic significance. However, little is known about their detailed functional roles in either free-living or parasitic nematodes. Methods: A genome-wide identification and curation were performed to screen the FAR family members of Haemonchus contortus. Their transcription patterns in worms were also analysed to identify the targets. Ligand binding assay and molecular docking were conducted to verify the fatty acid binding activities of FAR proteins of interest. RNA interference (RNAi) and heterologous expression (rescuing) experiments were designed to explore the potential roles of the selected FAR protein in nematodes. Localisation of the protein was shown in sections of paraffin-embedded worms after an immunohistochemistry (IHC) assay. Results: Here, an orthologue of far-6 in the model organism Caenorhabditis elegans (Ce-far-6) was functionally characterised in a parasitic nematode, H. contortus (Hc-far-6). It is demonstrated that knockdown of Ce-far-6 gene did not affect worm fat content, reproduction, or lifespan, but decreased worm body length at an early life stage of C. elegans. In particular, the Ce-far-6 mutant associated phenotype was completely rescued by Hc-far-6, suggesting a conserved functional role. Surprisingly, there were distinct tissue expression patterns of FAR-6 in the free-living C. elegans and parasitic H. contortus. High transcriptional level of Hc-far-6 and dominant expression of FAR-6 in the intestine of the parasitic stage of H. contortus link this gene/protein to nematode parasitism. Conclusions: These findings substantially enhance our understanding of far genes and the associated lipid biology of this important parasitic nematode at a molecular level, and the approaches established are readily applicable to the studies of far genes in a broad range of parasites. [ABSTRACT FROM AUTHOR]

Parasitic roundworms (nematodes) have lost genes involved in the de novo biosynthesis of haem, but have evolved the capacity to acquire and utilise exogenous haem from host animals. However, very little is known about the processes or mechanisms underlying haem acquisition and utilisation in parasites. Here, we reveal that HRG-1 is a conserved and unique haem transporter in a broad range of parasitic nematodes of socioeconomic importance, which enables haem uptake via intestinal cells, facilitates cellular haem utilisation through the endo-lysosomal system, and exhibits a conspicuous distribution at the basal laminae covering the alimentary tract, muscles and gonads. The broader tissue expression pattern of HRG-1 in Haemonchus contortus (barber's pole worm) compared with its orthologues in the free-living nematode Caenorhabditis elegans indicates critical involvement of this unique haem transporter in haem homeostasis in tissues and organs of the parasitic nematode. RNAi-mediated gene knockdown of hrg-1 resulted in sick and lethal phenotypes of infective larvae of H. contortus, which could only be rescued by supplementation of exogenous haem in the early developmental stage. Notably, the RNAi-treated infective larvae could not establish infection or survive in the mammalian host, suggesting an indispensable role of this haem transporter in the survival of this parasite. This study provides new insights into the haem biology of a parasitic nematode, demonstrates that haem acquisition by HRG-1 is essential for H. contortus survival and infection, and suggests that HRG-1 could be an intervention target candidate in a range of parasitic nematodes. Author summary: Parasitic nematodes cannot synthesise haem de novo and must exploit haem from the environment or host animals. Mechanisms securing haem acquisition and its systemic distribution in these pathogens remain to be elucidated. In the present study, we identify a unique haem transporter HRG-1 in a range of nematodes commonly found in animals, which shows haem binding activity in vitro, haem transporting activity in yeast, and an important role in haem homeostasis in the free-living model organism Caenorhabditis elegans. This transporter distributes in the basal laminae and interacts with a vacuolar ATPase in Haemonchus contortus (a paradigm and model to screen anthelmintic targets), enabling haem transport among tissues in this parasitic nematode. RNAi-mediated gene knockdown of hrg-1 results in decreased viability of the infective larvae of barber's pole worm, which cannot establish infection or survive in the host animal. The uniqueness and essentiality of HRG-1 in this parasitic nematode provide novel insights into the haem uptake and distribution in helminths and a target candidate for the control of nematode infection. [ABSTRACT FROM AUTHOR]

Toxocariasis is a neglected parasitic disease caused predominantly by larvae of Toxocara canis. While this zoonotic disease is of major importance in humans and canids, it can also affect a range of other mammalian hosts. It is known that mucins secreted by larvae play key roles in immune recognition and evasion, but very little is understood about the molecular interactions between host cells and T. canis. Here, using an integrative approach (affinity pull-down, mass spectrometry, co-immunoprecipitation and bioinformatics), we identified 219 proteins expressed by a murine macrophage cell line (RAW264.7) that interact with prokaryotically-expressed recombinant protein (rTc-MUC-1) representing the mucin Tc-MUC-1 present in the surface coat of infective larvae of T. canis. Protein-protein interactions between rTc-MUC-1 and an actin binding protein CFL1 as well as the fatty acid binding protein FABP5 of RAW264.7 macrophages were also demonstrated in a human embryonic kidney cell line (HEK 293T). By combing predicted structural information on the protein-protein interaction and functional knowledge of the related protein association networks, we inferred roles for Tc-MUC-1 protein in the regulation of actin cytoskeletal remodelling, and the migration and phagosome formation of macrophage cells. These molecular interactions now require verification in vivo. The experimental approach taken here should be readily applicable to comparative studies of other ascaridoid nematodes (e.g. T. cati, Anisakis simplex, Ascaris suum and Baylisascaris procyonis) whose larvae undergo tissue migration in accidental hosts, including humans. Author summary: Toxocariasis is a neglected parasitic disease of humans caused mainly by larvae of Toxocara canis. Given that T. canis is zoonotic and can infect a range of mammals, there has been substantial interest in host-parasite relationships, with studies showing that T. canis larvae secrete abundant mucins that effect/modulate immune responses and disease pathogenesis. To improve the understanding of immunomolecular interactions, we investigated the role(s) of the protein component of a mucin (Tc-MUC-1) secreted by infective larvae using a well-defined murine macrophage line (RAW264.7). The non-glycosylated recombinant protein of Tc-MUC-1 (designated rTc-MUC-1) was shown to interact with at least 219 proteins of RAW264.7 cells, particularly with the actin binding protein (CFL1) and a fatty acid binding protein (FABP5), which are involved in cell migration and phagocytosis, respectively. Based on these findings, we propose that Tc-MUC-1 regulates cytoskeletal organisation and signal transduction in host macrophages. It would be interesting to establish, using the integrative experimental approach employed here, whether the role(s) of Tc-MUC-1 protein homologues of related ascaridoids are conserved. [ABSTRACT FROM AUTHOR]

Simple Summary: Toxoplasmosis is one of the most common parasitic zoonoses in the world. It not only threatens human health and safety but also causes considerable losses to the global animal husbandry industry. Swine, as an intermediate host of Toxoplasma gondii, has important economic and public health significance, and the global prevalence of T. gondii in swine varies between 10% and 60%. Therefore, it is important to establish simple, effective, sensitive and specific diagnostic methods for the detection and prevention of T. gondii infection in swine. In the present study, we developed a colloidal gold immunochromatographic strip based on rTgROP14, recombinant protein A and monoclonal antibody TgROP14-5D5 for serological detection of T. gondii in swine populations. This new ICT achieved good specificity and sensitivity and has potential for routine serological detection of T. gondii in swine. Toxoplasma gondii, a worldwide distributed apicomplexan protozoan, can infect almost all warm-blooded animals and may cause toxoplasmosis. In order to provide a point-of-care detection method for T. gondii infection, an immunochromatographic test (ICT) was established. The proposed test uses recombinant T. gondii rhoptry protein 14 (ROP14) conjugated with 20 nm gold particles, recombinant protein A as the detection line and monoclonal antibody TgROP14-5D5 as the control line. The specificity, sensitivity, positive predictive value, negative predictive value and stability of this new ICT were evaluated. rTgROP14 was specifically recognized by positive serum of T. gondii but not negative serum. mAb TgROP14-5D5 showed higher specific recognition of T. gondii antigens and was therefore selected for subsequent colloidal gold strip construction. The new ICT based on TgROP14 exhibited good diagnostic performance with high specificity (86.9%) and sensitivity (90.9%) using IHA as a "reference standard". Among 436 field porcine sera, ICT and IHA detected 134 (30.7%) and 99 (22.7%) positive samples, respectively. The relative agreement was 87.8%. These data indicate that this new ICT based on TgROP14 is a suitable candidate for routine testing of T. gondii in the field. [ABSTRACT FROM AUTHOR]

Nuclear hormone receptors (NHRs) are emerging target candidates against nematode infection and resistance. However, there is a lack of comprehensive information on NHR-coding genes in parasitic nematodes. In this study, we curated the nhr gene family for 60 major parasitic nematodes from humans and animals. Compared with the free-living model organism Caenorhabditis elegans , a remarkable contraction of the nhr family was revealed in parasitic species, with genetic diversification and conservation unveiled among nematode Clades I (10–13), III (16–42), IV (33–35) and V (25–64). Using an in vitro biosystem, we demonstrated that 40 nhr genes in a blood-feeding nematode Haemonchus contortus (clade V; barber's pole worm) were responsive to host serum and one nhr gene (i.e. , nhr-64) was consistently stimulated by anthelmintics (i.e. , ivermectin, thiabendazole and levamisole); Using a high-throughput RNA interference platform, we knocked down 43 nhr genes of H. contortus and identified at least two genes that are required for the viability (i.e. , nhr-105) and development (i.e. , nhr-17) of the infective larvae of this parasitic nematode in vitro. Harnessing this preliminary functional atlas of nhr genes for H. contortus will prime the biological studies of this gene family in nematode genetics, infection, and anthelmintic metabolism within host animals, as well as the promising discovery of novel intervention targets. [Display omitted] • The first genetic landscape and functional atlas for the nhr gene family in parasitic nematodes. • nhr-64 is consistently stimulated by ivermectin, thiabendazole and levamisole. • nhr-17 and nhr-105 are required for larval viability in Haemonchus contortus. [ABSTRACT FROM AUTHOR]

Highlights: [•] The potent protection in mice immunized with recombinant protein ROP18 + Re was analyzed. [•] The rROP18 triggered a stronger humoral and cellular response against T. gondii. [•] Re is a promising vaccine adjuvant against toxoplasmosis, deserves further evaluation. [Copyright &y& Elsevier]

Toxoplasma gondii is a widespread and specialized intracellular protozoan pathogen that affects one third of the world' s population, posing a great threat to public health. As the definitive host, cats excrete oocysts and play a crucial role in the transmission of toxoplasmosis. The current diagnostic tools usually require bulky equipment and expertize, which hinders the efficient diagnosis and intervention of Toxoplasma infection in cats. In this study, we combined (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to establish an easier method for the detection of T. gondii oocysts in cat fecal samples. The sensitivity, specificity, and practicability of the established RPA-CRISPR/Cas9 method were evaluated using a lateral flow strip, with the limitation of detection determined at 10 plasmid copies/μL (corresponding to about one oocyst), cross reactivity to none of Giardia lamblia , Cryptosporidium sp., Microsporidium biberi and Blastocystis hominis that also commonly found in cats, and comparable performance in detecting T. gondii in clinical samples to conventional PCR amplification. This RPA-CRISPR/Cas9 method provides an alternative to conventional molecular tools used in the clinical diagnosis of Toxoplasma infection in cats and other animals. • The detection limit of RPA-CRISPR/Cas9 method is 10 plasmid copies/μL. • It has no cross-reactivity to Giardia lamblia , Cryptosporidium sp., Microsporidium biberi and Blastocystis hominis. • It has comparable performance in detecting T. gondii in clinical cat fecal samples to conventional PCR amplification. [ABSTRACT FROM AUTHOR]

[Display omitted] • Haemonchus contortus protein Hc -SPI-I8A functions as an inhibitor of blood clotting contributed by the TIL domain. • Hc -SPI-I8 is highly expressed in the parasitic stages and at the hypodermis, intestine and gonads of H. contortus. • Hc -SPI-I8 was in the same clade as the homologues, which could inhibit coagulation by interacting with clotting factors. • Hc -SPI-I8A is most likely disturbing the extrinsic coagulation cascade by interacting with Oa TSP1CP through its TIL domain. • Hc -SPI-I8A might also interfering in the intrinsic coagulation cascade by an unknown mechanism. Haemonchus contortus , a blood-feeding nematode, inhibits blood coagulation at the site of infection to facilitate blood-sucking and digesting for successful parasitism. However, the mechanism underlying anti-coagulation at the host-parasite interface is largely unknown. In the current study, Hc-spi-i8 , which has two greatly different transcripts named Hc-spi-i8a and Hc-spi-i8b , respectively, was described. Hc -SPI-I8A was a serine protease inhibitor containing a trypsin inhibitor-like cysteine rich (TIL) domain, while Hc -SPI-I8B was not. Hc -SPI-I8A/B were primarily expressed in the hypodermis, intestines and gonads in the parasitic stages of H. contortus. Hc -SPI-I8A interacted with Ovis aries TSP1-containing protein (Oa TSP1CP), which was determined by yeast two-hybrid, co-immunoprecipitation (Co-IP), pull down and co-localization experiments. The blood clotting time contributed by the TIL domain was prolonged by Hc -SPI-I8A. Hc -SPI-I8A is most likely interfering in the extrinsic coagulation cascade by interacting with Oa TSP1CP through its TIL domain and intrinsic coagulation cascade by an unknown mechanism. These findings depict a crucial point in the host-parasite interaction during H. contortus colonization, which should contribute to drug discovery and vaccine development in fighting against this important parasite worldwide. [ABSTRACT FROM AUTHOR]

The high incidence and severe damage caused by Toxoplasma gondii infection clearly indicates the need for the development of a vaccine. In this study, we evaluated the immune responses and protection against toxoplasmosis by immunizing ICR mice with a multiantigenic DNA vaccine. To develop the multiantigenic vaccine, two T. gondii antigens, MIC3 and ROP18, selected on the basis of previous studies were chosen. ICR mice were immunized subcutaneously with PBS, empty pcDNA3.1 vector, pMIC3, pROP18, and pROP18-MIC3, respectively. The results of lymphocyte proliferation assay, cytokine, and antibody determinations showed that mice immunized with pROP18-MIC3 elicited stronger humoral and Th1-type cellular immune responses than those immunized with single-gene plasmids, empty plasmid, or phosphate-buffered saline. After a lethal challenge with the highly virulent T. gondii RH strain, a prolonged survival time in pROP18-MIC3-immunized mice was observed in comparison to control groups. Our study indicates that the introduction of multiantigenic DNA vaccine is more powerful and efficient than single-gene vaccine, and deserves further evaluation and development. [ABSTRACT FROM AUTHOR]

Hypobiosis (facultative developmental arrest) is the most important life-cycle adaptation ensuring survival of parasitic nematodes under adverse conditions. Little is known about such survival mechanisms, although ascarosides (ascarylose with fatty acid-derived side chains) have been reported to mediate the formation of dauer larvae in the free-living nematode Caenorhabditis elegans. Here, we investigated the role of a key gene acox-1, in the larval development of Haemonchus contortus, one of the most important parasitic nematodes that employ hypobiosis as a routine survival mechanism. In this parasite, acox-1 encodes three proteins (ACOXs) that all show a fatty acid oxidation activity in vitro and in vivo, and interact with a peroxin PEX-5 in peroxisomes. In particular, a peroxisomal targeting signal type1 (PTS1) sequence is required for ACOX-1 to be recognised by PEX-5. Analyses on developmental transcription and tissue expression show that acox-1 is predominantly expressed in the intestine and hypodermis of H. contortus, particularly in the early larval stages in the environment and the arrested fourth larval stage within host animals. Knockdown of acox-1 and pex-5 in parasitic H. contortus shows that these genes play essential roles in the post-embryonic larval development and likely in the facultative arrest of this species. A comprehensive understanding of these genes and the associated β-oxidation cycle of fatty acids should provide novel insights into the developmental regulation of parasitic nematodes, and into the discovery of novel interventions for species of socioeconomic importance. Author summary: Haemonchus contortus is one of the most pathogenic nematodes causing substantial economic losses worldwide that undergo facultative arrest (diapause) to survive harsh environmental and host conditions. However, the molecular basis (e.g., fatty acid oxidation and dauer pheromone synthesis) underlying such survival mechanisms has not yet been elucidated in parasitic nematodes. Here, we show that an acyl-CoA oxidase ACOX-1 interacts with a peroxin PEX-5 and functions in fatty acid oxidation in peroxisomes. Further, knockdown of ACOX-1 and PEX-5 results in a lethal phenotype of the infective stage and likely developmental parasitic larval stage within host animals. This data demonstrates an essential role of ACOX-1-PEX-5 and potentially ascarosides signalling in the facultative arrested larval development of parasitic nematodes, and suggests possible intervention targets. [ABSTRACT FROM AUTHOR]

Protease inhibitors are major components of excretory/secretory products released by parasitic nematodes and have been proposed to play roles in host-parasite interactions. Haemonchus contortus (the barber's pole worm) encodes for several serine protease inhibitors, and in a previous study we identified a trypsin inhibitor-like serine protease inhibitor of this blood-feeding nematode, SPI-I8, as necessary for anticoagulation. Here, we demonstrated that a bovine pancreatic trypsin inhibitor/Kunitz-type serine protease inhibitor (BPTI/Kunitz) domain-containing protein highly expressed in parasitic stages, HCON_00133150, is involved in suppressing proinflammatory cytokine production in mammalian cells. Fluorescent labelling of HCON_00133150 revealed a punctate localisation at the inner hypodermal membrane of H. contortus , an organ closely related to the excretory column. Yeast two-hybrid screening and immunoprecipitation-mass spectrometry identified that the recombinant HCON_00133150 physically interacted with a range of host proteins including the G protein subunit beta 1 of sheep (Ovis aries ; Oa GNB1), a negative regulator of NLRP3 inflammasome activation. Interestingly, heterologous expression of HCON_00133150 enhanced the inhibitory effect of Oa GNB1 on NLRP3 inflammasome and the maturation of proinflammatory cytokines IL-1β and IL-18 in transfected cells. 1-to-1 orthologues (n = 33) of BPTI/Kunitz inhibitor domain-containing proteins were predicted in clades III, IV and V (but not clade I) parasitic nematodes. Structural (tandem BPTI/Kunitz inhibitor domains inverted into the globular reticulation) and functional (a GNB1 enhancer) characterisation of HCON_00133150 and its orthologues elucidated that these molecules might contribute to immune suppression by parasitic nematodes in animals and humans. • HCON_00133150 encodes a collagen-like BPTI/Kunitz domain-containing protein. • More than 700 proteins of sheep PBMCs and abomasum interact with HCON_00133150. • HCON_00133150 inhibits IL-1β/IL-18 maturation in vitro by interacting with GNB1. [ABSTRACT FROM AUTHOR]

• Haemonchus contortus protein Hc-DHS-28 has 3-hydroxyacyl CoA dehydrogenase activity. • Hc-dhs-28 is highly transcribed in the diapause worm and located in the peroxisome. • Hc-dhs-28 shares high similarity in gene function with Caenorhabditis elegans (Ce) -dhs-28. • Hc-dhs-28 might regulate the formation of diapause in H. contortus. • Immunisation with recombinant Hc-DHS-28 could protect the host after H. contortus infection. Haemonchus contortus could enter the diapause stage to avoid hostile conditions, however the inducing mechanism still remains poorly understood. A similar dauer strategy exists in Caenorhabditis elegans , and dauer phenomones, which are produced through a four step cycle of peroxisomal fatty acid β-oxidation, are essential in this stage. In this study, a novel gene, Hc-dhs-28 , was identified and characterised. Hc-DHS-28 was the homologue of Ce-DHS-28, a key enzyme in the oxidation cycle, and the protein contained a short chain dehydrogenase domain and a peroxisomal targeting signal 1. The expression pattern of Hc-DHS-28 detected by quantitative real-time PCR and indirect immunofluorescence assay revealed that this protein was mainly expressed in the intestine and subdermal regions of larvae at diapause and in free-living stages. Enzyme activity analysis confirmed its 3-hydroxyacyl CoA dehydrogenase activity with 121, 149, 162 and 166 as key functional sites; meanwhile co-localization in human embryonic kidney 293 cells indicated that Hc-DHS-28 was targeted to the peroxisome of cytoplasm under the guide of peroxisomal targeting signal 1, which was consistent with the functional domain prediction of Hc-dhs-28. Overexpression, rescue and RNA interference experiments were carried out to explore the function of Hc-dhs-28. Our results showed that Hc-dhs-28 was very similar to Ce-dhs-28 and partially rescued its function in C. elegans. RNAi with Hc-dhs-28 in C. elegans led to decreased transcription of genes in the peroxisomal fatty acid β-oxidation cycle, considerable fat accumulation and dauer formation defects. Furthermore, immunisation with recombinant Hc-DHS-28 protein in sheep was able to maintain the body weight of the host after infection and reduce the worm burden. In conclusion, Hc-DHS-28 is most likely involved in the peroxisome fatty acid β-oxidation as the third 3-hydroxyacyl CoA dehydrogenase to regulate the production of diapause-related pheromones, and then influence the formation of diapause in H. contortus. [ABSTRACT FROM AUTHOR]

Background: Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods: HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results: γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions: Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival. [ABSTRACT FROM AUTHOR]

Excretory/secretory proteins of Haemonchus contortus (HcESPs) intermingle comprehensively with host immune cells and modulate host immune responses. In this study, H contortus ES antigen named as elongation factor 1 alpha (HcEF‐1α) was cloned and expressed. The influences of recombinant HcEF‐1α on multiple functions of goat peripheral blood mononuclear cells (PBMCs) were observed in vitro. Immunoblot analysis revealed that rHcEF‐1α was recognized by the serum of goat infected with H contortus. Immunofluorescence analysis indicated that rHcEF‐1α was bound on surface of PBMCs. Moreover, the productions of IL‐4, TGF‐β1, IFN‐γ and IL‐17 of cells were significantly modulated by the incubation with rHcEF‐1α. The production of interleukin IL‐10 was decreased. Cell migration, cell proliferation and cell apoptosis were significantly increased; however, nitric oxide production (NO) was significantly decreased. The MHC II molecule expression of cells incubated with rHcEF‐1α was increased significantly, whereas MHC‐I was not changed as compared to the control groups (PBS control and pET32a). These findings indicated that rHcEF‐1α protein might play essential roles in functional regulations of HcESPs on goat PBMC and mediate the immune responses of the host during host‐parasite relationship. [ABSTRACT FROM AUTHOR]

Both Fasciola hepatica and Clonorchis sinensis are endemic in China, South Korea, Japan and other Southeast Asian countries. Reliable and sensitive diagnostic methods are needed for detecting their infections in humans and animals. Differential simplex and duplex polymerase chain reaction (PCR) methods were developed. The PCRs targeted the second internal transcribed spacer ( its 2) (408 bp) of F. hepatica , and NADH dehydrogenase subunit 2 gene ( nad 2) (527 bp) of C. sinensis . Both simplex PCRs detected as little as 2 pg genomic DNA in one microliter in a 25 μL PCR reaction system. The duplex PCR had similar detection limit as well, and detected as low as one egg in 200 mg feces. These methods were analytical specific with no amplification being observed from the gemonic DNA of Fasciolopsis buski , Haemonchus contortus , Ascaris ovis or Eimeri ahsata . Of 158 sheep fecal samples collected from various farms, four and one samples were PCR-positive for F. hepatica and C. sinensis , respectively. The duplex PCR method described here is time-saving and convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of F. hepatica and C. sinensis in endemic area. [ABSTRACT FROM AUTHOR]

Citrinin (CIT) is a hepato-nephrotoxic fungal metabolite produced by the genera Penicillium, Aspergillus and Monascu. There is an increasing demand for rapid and economical methods for detection CIT residues in fruit. In this study, we developed an immunochromatographic strip (ICS) for detection of citrinin (CIT) residues in fruit for the first time. Anti-CIT monoclonal antibody (McAb) 2B9 was prepared, with a binding affinity of 9.39 × 108 L/moL. Conjugates CIT-BSA and McAb 2B9 were used to develop the ICS which could be completed in 5 min, with the detection limit of 50 ng/mL and no cross reactivity with other mycotoxins. Analysis of CIT in 64 fruit samples revealed that data obtained from the ICS test were in good agreement with indirect competitive enzyme-linked immunosorbent assays (ic-ELISAs) and high performance liquid chromatography (HPLC). This result demonstrated that the ICS test could be used as a rapid, reliable, cost-effective and user-friendly qualitative tool for detection of CIT residues on-site. [ABSTRACT FROM AUTHOR]

The immunochromatographic test (ICT) is a convenient and low-cost method that can rapidly obtain results (10 min) under normal conditions. In this study, we established an ICT assay with two monoclonal antibodies: TgSAG3-3A7 and TgSAG3-4D5 based on the conserved protein of TgSAG3 that can be expressed in all the infective stages of T. gondii. 20 nm gold particles were prepared and conjugated with TgSAG3-3A7 MAb at the concentration of 12.5 μg/mL. TgSAG3-4D5 MAb were used as the capture antibody because of its higher affinity tested by ELISA. The detection limit of the ICT assay was 100 ng with visual detection under optimal conditions of analysis. Positive porcine serum of Cryptosporidium suis , Mycoplasma suis , Streptococcus suis , Salmonella choleraesuis , Cysticercus cellulosae , Isospora suis , and Trichinella spiralis were used to evaluate the specificity of this ICT and no cross-reactivity was observed. 310 porcine serum samples obtained from pig farms in Zhejiang Province, China were detected by this ICT and ELISA kit, 23 positive samples were found by the developed strip with the rate of 7.42% comparing with 22 positive samples detected by the commercially ELISA kit which the positive rate was 7.1%, the relative sensitivity and specificity of this ICT are 100% and 99.65%. Therefore, the ICT established in this study is proved effective, simple, specific and sensitive of T. gondii detection. [ABSTRACT FROM AUTHOR]

RAMP60 , RAMP70 , RAMP80 and RAMPtp extracted from the rhizome of Atractylodis macrocephalae Koidz . were chemically analyzed and tested for their lymphocyte stimulation. Among them, only RAMPtp was found to induce proliferation of lymphocytes from bovine supramammary lymph node (SMLN). It was found that RAMPtp was homogeneous polysaccharide with Mn of 1.867 × 10 3 by GPC analysis, composed of glucose, mannose, rhamnose, arabinose and galactose, with mass percentages of 60.67%, 14.99%, 10.61%, 8.83% and 4.90%, respectively by HPLC analysis, connected by 1, 3-linked β-D Galp and 1, 6-linked β-D Galp residues by NMR analysis and was amorphous in structure by SEM analysis. When SMLN lymphocytes were cultured with RAMPtp in vitro , increased [Ca 2+ ] i , more cells in S and G2/M phases and upregulated IFN-γ and IL-17A while downregulated IL-4 mRNA expressions as well as the binding between SMLN lymphocytes and RAMPtp were observed. These findings might explain the lymphocyte-stimulating property of RAMPtp . [ABSTRACT FROM AUTHOR]

Toxoplasma gondii ( T. gondii ) is one of the most common parasite that can infect almost any warm-blooded animals including humans. The cyclic nucleotide-dependent protein kinase (PKA) regulates a spectrum of intracellular signal pathways in many organisms. Protein kinase catalytic subunit (PKAC) is the core of the whole protein, and plays an important role in the life cycle of T.gondii . Here, T.gondii PKAC ( Tg PKAC) overexpression strain ( Tg PKAC-OE) was constructed. The growth of the Tg PKAC-OE, RH △Ku80 , and Tg PKAC inhibition strains ( Tg PKAC-H89) were analysed by SYBR-green real-time PCR, and the ultrastructure was observed by transmission electron microscopy. The survival rate in mice was also recorded to analyse the virulence of the parasites. We also investigated the subcellular localization of Tg PKAC in Vero cells by laser scanning microscope. We found that Tg PKAC-OE strain exhibited obviously increased growth rate in Vero cells in vitro , and infected mice survived for a shorter time compared to wild type strain. Ultrastructural analysis found more autophagosomes-like structures in Tg PKAC-H89 parasite compared to RH △Ku80 strain, and the relative expression level of Toxoplasma gondii autophagy-related protein (ATG8) in Tg PKAC-H89 parasite was higher than wild type parasite. Laser confocal results showed that Tg PKAC was mainly expressed in the cytoplasm of Vero cells. In conclusion, we hypothesized that inhibition of Tg PKAC could cause autophagy of Toxoplasma gondii and then influence the replication of the parasite. Tg PKAC plays an important role in parasite virulence in vivo , and the subcellular localization was successfully detected in Vero cells. Our data will provide a basis for further study of Tg PKAC function and help screen drug targets of T. gondii . [ABSTRACT FROM AUTHOR]

The apicoplast, which is the result of secondary endosymbiosis, is a distinctive subcellular organelle and a crucial therapeutic target for apicomplexan parasites. The majority of apicoplast-resident proteins are encoded by the nuclear genome and target the apicoplast via bipartite targeting signals consisting of a signal peptide and a transit peptide. The properties and functions of these peptides are poorly understood, which hinders the identification of apicoplast proteins and the study for plastid evolution. Here, the targeting signals of the recently discovered apicoplast tRNA thiouridylase Tg MnmA of Toxoplasma gondii were analyzed. Our data using a reporter (the enhanced green fluorescent protein) fused with individual fragments containing various numbers of its N-terminal amino acids unequivocally revealed that the first 28 amino acids of Tg MnmA functioned as a signal peptide for cellular secretion. The N-terminal 150 amino acids were sufficient to direct the fusion protein to the apicoplast, whereas its deletion caused the fusion protein to be localized to the mitochondrion. Our data further demonstrated that the apicoplast, rhoptry, and mitochondrion shared similar targeting signals, indicating that the apicoplast localization peptide was trans-organellar in function. In addition, the apicoplast localization peptide was important for the healthy proliferation of tachyzoites. In conclusion, the targeting signals of the nucleus-encoded apicoplast-targeted protein Tg MnmA have been mapped out and the importance of this localization peptide has been elucidated in the current study. • The first N-terminal 150 AA of Tg MnmA target the EGFP to the apicoplast. • Without the localization peptide, Tg MnmAΔ1−150-EGFP localizes to the mitochondrion. • Mis-localization of Tg MnmA greatly affects the proliferation of T. gondii in vitro. [ABSTRACT FROM AUTHOR]

Echinococcus granulosus is the causative pathogen of cystic echinococcosis, a serious disease endangering human and animal health. In this study, an immunochromatographic strip was developed based on the recombinant protein Heat shock protein 70 (HSP70) for the serological detection of E. granulosus . The protocol completes within 20 min requiring no specialized equipment or chemical reagents, while specificity tests confirmed no cross-reactivity with positive serum of Fasciola hepatica , Haemonchus contortus , Peste des petits ruminants virus (PPRV) and Foot-and-mouth disease virus (FMDV). The strips remained stable after storage at 4 °C for up to 8 months. Both immunochromatographic strip and ELISA tests were applied to detect E. granulosus antibody in a total of 728 serum samples obtained from slaughter houses in Zhejiang Province. Our data revealed positive rates of 2.61 and 1.65% by immunochromatographic strip and ELISA methods, respectively. The immunochromatographic strip test developed in this study provides a simple, specific and rapid method of E. granulosus antibody detection and infected sheep monitoring. [ABSTRACT FROM AUTHOR]

The parasite Cryptosporidium baileyi can infect the larynx, trachea, bursa and cloaca of poultry, causing high mortality during severe infection and leading to substantial economic losses of the poultry industry. The rhomboid protein is very important in Cryptosporidium infection. In this study, a nucleic acid based vaccine candidate pEGFP-CbROM was constructed. After orally challenging with C. baileyi oocysts, the corresponding immune responses induced were analyzed and the immunoprotective effect evaluated in chickens. Obtained results revealed that this nucleic acid based vaccine could induce antibody responses and peripheral blood T lymphocytes proliferation significantly ( P < 0.05), while the peripheral blood B lymphocyte proliferation increased significantly ( P < 0.05) only at a high dose of 100 μg of pEGFP-CbROM, compared with the PBS control group. After C. baileyi infection, the duration of oocysts shedding was shortened by 2 days in the 100 μg pEGFP-CbROM group, and the rate of reduction could reach to around 71.3%. While no significant difference in body weight gain was observed among the immunized groups ( P > 0.05), the differences between the immunized and the non-immunized groups were found to be significant ( P < 0.05). Our data provides a useful basis for further work in cryptosporidiosis prevention and treatment. [ABSTRACT FROM AUTHOR]

Babesia spp. are apicomplexan protozoan parasites of the red blood cells of mammals and are transmitted by ticks. Bovine babesiosis mainly caused by Babesia bovis and Babesia bigemina occurs worldwide, which is a great threat to animal health. Microscopy examination is a gold standard for the diagnosis of babesiosis. However, its sensitivity is too low. This study was conducted to establish a simple, efficient and fast LAMP-LFP method used for early diagnosis of animal babesiosis. LAMP was developed with a set of four primers targeting and amplifying six distinct regions of cytochrome b gene of Babesia spp. under isothermal conditions. Afterwards, a chromatographic lateral-flow dipstick (LFD) was used to detect LAMP products that were labeled with FITC at the 5′ end, avoiding gel electrophoresis. The LAMP-LFD method was very specific, yielding no positive results with DNA templates of Theileria sergenti , Thenileria ovis , Theileria equi and Toxoplasma gondii . The LAMP-LFP was highly sensitive and could detect 0.85 fg B. bigemina DNA and 0.14 fg B. bovis DNA, 100-fold higher than a conventional PCR assay. This method could be adapted for quick and accurate diagnosis of bovine babesiosis in the fields in case the whole blood could be directly used, especially for identifying carrier animals with very low parasitaemia. [ABSTRACT FROM AUTHOR]

Single chain variable fragments (scFvs) against citrinin (CIT) were selected from a scFv library constructed from the splenocytes of non-immunized mice by an improved eukaryotic ribosome display technology in this study. Bovine serum albumin (BSA)/ CIT-BSA and ovalbumin (OVA)/ CIT-OVA were used as the antigens to select specific anti-CIT scFvs. Eukaryotic in situ RT-PCR method was used to recover the selected mRNA after every affinity selection. After six rounds of ribosome display, expression vector pTIG-TRX carrying specific scFv DNAs were constructed and transformed into Escherichia coli BL21 (DE3) for protein expression. Thirteen positive clones were selected out of which three (designated 23, 68 and 109) showed high binding activity and specificity to CIT by indirect ELISA, while no clone showed binding activity with carrier proteins. The three scFvs showed high specificity to CIT and the cross reactivity with other mycotoxins was below 0.01% as determined by indirect competitive ELISA. These specific scFvs offer a potential novel immunoassay method for CIT residues. This study confirmed the effectiveness of the improved eukaryotic ribosome display system and could be used as a reference for the selection of scFvs specific to other small molecules using ribosome display. [ABSTRACT FROM AUTHOR]

Infective Haemonchus contortus larvae (L3s) are able to protect themselves from desiccation. To explore the molecular mechanisms of desiccation survival, mRNA differential display RT-PCR was used to screen differentially expressed genes in L3s upon desiccation, followed by RNAi experiments to define gene functions. In this, 58 differentially expressed transcripts were obtained. Among these, the BF-U01A and CH-U02A fragments represent genes with the highest identity percentage in bioinformatic analysis. They were named Hc-ubq and Hc-gst based on their respective homologous ubiquitin in Caenorhabditis elegans and glutathione S-transferase in H. contortus . Quantitive RT-PCR results indicated that they were both up-regulated in desiccated L3s. Hc-ubq and Hc-gst RNAi in H. contortus showed reduced survival rate of L3s, with unchanged locomotion behavior. Homologous Ce-ubq-2 and Ce-gst-7 RNAi in C. elegans also displayed higher larval death rate. These results suggest that ubq and gst may play important roles in nematode desiccation tolerance. Our study analyzed desiccation resistance related genes in H. contortus L3s, and revealed significant research implications on the mechanisms behind nematode desiccation survival. [ABSTRACT FROM AUTHOR]

The loop-mediated isothermal amplification (LAMP) assay is a novel method that rapidly amplifies DNA with high specificity and sensitivity under isothermal conditions. In this study, we established a LAMP assay with six primers targeting a highly conserved region of Toxoplasma gondii ITS-1 sequence. The amplification protocol completes within 30 min under isothermal condition in a 65 °C water bath while specificity tests confirmed no cross-reactivity with DNA templates of Neospora caninum , Eimeria tenella , Cryptosporidium parvum , Listeria monocytogenes and Streptococcus suis . The detection limit of the LAMP assay was 0.9 fg T. gondii genomic DNA, a sensitivity that was 10-fold higher than that of a conventional PCR assay. Both LAMP assay and conventional PCR were applied to detect T. gondii genomic DNA in 118 diaphragm samples obtained from pig farms in Zhejiang Province, China. Our results showed that the LAMP assay is more sensitive than conventional PCR (13.56% and 9.32%). The LAMP assay established in this study provides a simple, specific, sensitive and rapid method of T. gondii genomic DNA detection, hence is expected to plays an important role in the monitoring of T. gondii contamination in various food products. [ABSTRACT FROM AUTHOR]

Diapause induced in the early fourth stage of Haemonchus contortus is a strategy to adapt this nematode to hostile environmental conditions. In this study, we identified a new gene, Hc-fau , a homologue of human fau and Caenorhabditis elegans Ce-rps30 . Hc-fau encodes two proteins through alternative RNA splicing, Hc -FAUA and Hc -FAUB, consisting of 130 and 107 amino acids, respectively. Hc -FAU possesses a diverged ubiquitin-like (UBiL) protein domain and a conserved ribosome protein S30 domain. The protein is ubiquitously expressed, except in the gonad. However Hc-fau transcripts decrease significantly in diapausing L4s of H. contortus . In C. elegans , knockdown of Ce-rps30 confers an extended lifespan, increased lipid storage in the intestine and shortened body length. These morphological characteristics are comparable with dauer larvae of C. elegans , in which the gonad is condensed considerably. In contrast, a shortened lifespan is observed in C. elegans over-expressing Hc-faua , and especially Hc-faub , with hatching failure detected. The genes of insulin/IGF-1 signalling (IIS), TGF-β, cGMP, dafachronic acid (DA), apoptosis (AP) and fatty acids (FA) metabolism are all down-regulated in Ce-rps30 RNAi (RNA interference) worms, except for akt-1 and daf-16 . However, daf-16 up-regulation is inconsistent with its target gene down-regulation and the result from a heat stress assay in these worms. Daf-16 RNAi conducted in Ce-rps30 (tm6034/nt1) mutants failed to rescue the worms. The S30 domain stays in the nucleus, while UBiL accumulates in the cytoplasm. Compared with Hc -FAUA, results of UBiL domain and S30 domain over-expression indicate synergism between UBiL and S30 in regulating lifespan and reproduction. These results suggest the potential functions of Hc-fau in regulating larval diapause in H. contortus . [ABSTRACT FROM AUTHOR]

Abstract: Real-time PCR-based detection of Toxoplasma gondii is very sensitive and convenient for diagnosing toxoplasmosis. However, the performance of the PCR assays could be influenced by the target gene chosen. Here we evaluate a real-time PCR assay using double-stranded DNA dyes (SYBR® Green I assay) with a new set of primers targeting the SAG1 gene for the fast and specific detection of T. gondii. The assay showed higher sensitivity than conventional PCR protocols using T. gondii DNA as template. The detection limit of the developed real-time PCR assay was in the order of 1 tachyzoite. The assay was also assessed by experimentally infected mice and showed positive results for blood (25%), spleen (50%) and lung (50%) as early as 1 dpi. The specificity of the assay was confirmed by using DNA from Neospora caninum, Escherichia coli, Babesia bovis, Trypanosoma brucei, Cryptosporidium parvum, and Toxocara canis. Assay applicability was successfully tested in blood samples collected from slaughtered pigs. These results indicate that, based on SYBR® green I, the quantitative SAG1 assay may also be useful in the study of the pathogenicity, immunoprophylaxis, and treatment of T. gondii. [Copyright &y& Elsevier]

Abstract: A fast, sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for the detection of Toxoplasma gondii (T. gondii) in pork was developed. In this study, we used a conserved sequence of 18s rRNA of Toxoplasma gondii to design primers for RT-LAMP test. The amplication was able to finish in 60min under isothermal condition at 63°C by employing a set of six primers. The assay showed higher sensitivity than RT-PCR using T. gondii RNA as template. The RT-LAMP assay was also assessed for specificity and was found to precisely discriminate between positive and negative test samples. Furthermore, the assay correctly detected T. gondii from contaminated pork, and had the detect limit of 1 tachyzoite in 1g pork. This is the first report of a study which applied the RT-LAMP method to detect T. gondii from pork. As RT-LAMP requires very basic instruments and the results can be obtained by visual observation, this technique provides a simple and reliable tool for inspecting food which are T. gondii-contaminated. [Copyright &y& Elsevier]

Abstract: Toxoplasma gondii, capable of infecting all warm blooded animals, is one of the most successful parasites worldwide. It was reported that the single-gene vaccine with SAG1 or MIC3 could only produce partial protection against T. gondii and multiantigenic vaccines were more effective than single ones. In the present study, a multiantigenic DNA vaccine delivered by attenuated Salmonella typhimurium (ZJ111/pSAG1-MIC3) was constructed, which expresses surface protein SAG1 and micronemal protein MIC3. The safety and stability of ZJ111/pSAG1-MIC3 were evaluated and immune response with ZJ111/pSAG1 and ZJ111/pMIC3 were compared. The results of lymphocyte proliferation assay, antibody and cytokine determination show that mice immunized with ZJ111/pSAG1-MIC3 elicited stronger humoral and Th1-type cellular immune responses than other groups. The mice immunized with ZJ111/pSAG1-MIC3 also exhibited significant higher survival time after challenged with T. gondii RH strain. The current study shows that the oral multiantigenic DNA vaccine, ZJ111/pSAG1-MIC3, produces partial protection against T. gondii challenge. [Copyright &y& Elsevier]

Abstract: Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The T. gondii surface antigen protein SAG1 is a significant candidate vaccine against toxoplasmosis. In this study, we evaluated safety, stability of ZJ111/pcDNA3-SAG1, a DNA vaccine delivered in attenuated Salmonella typhimurium, and immune responses induced by immunizing ICR mice orally with ZJ111/pcDNA3-SAG1 of different doses. Mice had no significant differences in body weight between the groups before immunization and at week 4 after the booster immunization. The ZJ111/pcDNA3-SAG1 was eventually eliminated from the spleen and liver on week 6 post-immunization. The plasmid pcDNA3-SAG1 was stably maintained over 90% of the attenuated S. typhimurium population after 100 generations of growth in antibiotic-free media. Oral immunization of mice with ZJ111/pcDNA3-SAG1 elicited specific humoral responses and stimulated proliferation of splenocytes (P <0.05). The cellular immune response was associated with the production of IFN-γ, indicating that a Th-1 type response was elicited, which was confirmed by the production of large amounts of IgG2a (P <0.01). Mice immunized with ZJ111/pcDNA3-SAG1 displayed significant protection against an intraperitoneally challenge with 500 tachyzoite forms of T. gondii RH strain. Vaccination at 107 and 108 CFU per mice provided a 20% and 10% survival rate comparing 100% mortality of the non-immunized mice, exhibiting longer living time and better survival rate. These results confirmed a DNA vaccine delivered in attenuated S. typhimurium, ZJ111/pcDNA3-SAG1, can elicit specific immune response as well as provide effective protection against T. gondii infection, and the dosage of 107 CFU was a most considerate one. [Copyright &y& Elsevier]

• SYBR Green real-time PCR based on copro-DNA was developed for the detection of fasciolosis in sheep feces. • Real-time PCR in laboratory contributed to improving of sensitivity for fasciolosis. • New method for a cross-sectional study showed a higher efficiency. Fasciolosis is commonly diagnosed by microscopic detection of egg following sedimentation. However, this technique is time-consuming when a large number of samples must be processed and requires sufficient experience. Quantitative real-time PCR based on the detection of liver fluke ribosomal DNA in feces has been introduced, which is more accurate and liable to reflect the presence of flukes in hosts. This study aimed to develop an efficient molecular detection method in laboratory diagnosis. A cross-sectional study of 250 sheep was performed to detect Fasciola hepatica infections using gold standard microscopic detection, conventional PCR and real-time PCR. Both conventional and real-time PCRs targeted the internal transcribed spacer 2 (ITS-2). A composite reference standard(CRS) was used to analyze the sensitivity and specificity of three methods. Furthermore, the minimal amount of plasmid DNA detected by the real-time PCR was 1.67 pg plasmid DNA (equivalent to 1.1 × 106 copies). In conclusion, a highly sensitive and specific method for fasciolosis in sheep was developed. [ABSTRACT FROM AUTHOR]

Background: Pattern recognition receptors (PRRs) can recognize pathogen-associated molecular patterns and activate downstream signalling pathways, resulting in modulation of host immunity against pathogens. Here, we investigated whether PRR-mediated recognition is involved in host immune responses to the blood-feeding nematode Haemonchus contortus. Methods: During blood-feeding, H. contortus secretes immune-modulating antigens into host blood. Therefore, we stimulated sheep peripheral blood mononuclear cells (PBMCs) with H. contortus soluble extract (HcAg) and performed transcriptional profiling. Results: HcAg upregulated two genetically linked CLRs (CLEC2L and KLRG2), two NLRs attenuating inflammation (NLRP12 and NLRC3) and one G protein-coupled receptor with potent anti-inflammatory effects (HCAR2). Furthermore, several Th2-related transcription factors (ATF3, IRF4, BCL3 and NFATC) were also upregulated, which may confer anti-inflammatory type 2 immune responses to HcAg. Conclusions: Together, our preliminary studies provide new insights into how the host innate immune system controls type 2 immunity to H. contortus. Further work will be needed to identify H. contortus products recognized by the host innate immune system and determine the Th2 polarization ability of these putative PRR ligands. [ABSTRACT FROM AUTHOR]

Background: The strongylid parasite Haemonchus contortus causes severe anemia in domestic animals worldwide. Effective preventive and therapeutical agents are lacking, because of drug resistance and that little is known about the molecular mechanism of the interaction between H. contortus and host cells. Methods: A new gene, Hc-clec-160 , was discovered with RT-PCR. Transcriptional levels of Hc-clec-160 and Ce-clec-160 throughout different growth phases of corresponding nematodes were assayed by qPCR. Immunofluorescence staining of paraffin section were performed to determine the protein localization in adult worms of H. contortus. To monitor the promoter capacity of the 5'-flanking region of Ce-clec-160 , micro-injection was used. Overexpression and RNAi constructs was carried out in the N2 strain of Caenorhabditis elegans to find out the gene function of Hc-clec-160. Results: The full-length cDNA of 1224 bp of Hc-clec-160 was cloned by RT-PCR. The corresponding gene contained twelve exons. Its transcripts peaked in male adult worms. Hc-CLEC-160 was predicted to have a Willebrand factor type A (vWA) domain and a C-type lectin domain. The proteins were not detected by expression in C. elegans or paraffin section experiments in adult of H. contortus. Knockdown of Ce-clec-160 expression in C. elegans by RNAi resulted in shortened body length and decreased brood size. Conclusions: In this experiment, a new gene Hc-clec-160 was obtained in H. contortus and its function was addressed using a model organism: C. elegans. Our study showed that Hc-clec-160 possesses characteristics similar to those of Ce-clec-160 and plays an important role in the growth and reproduction of this parasitic nematode. [ABSTRACT FROM AUTHOR]