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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]

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]

Toxoplasma gondii is an obligate intracellular apicomplexan parasite that infects almost all warm-blooded vertebrates. Heat shock proteins (HSP) regulate key signal transduction events in many organisms, and heat shock protein 90 (Hsp90) plays an important role in growth, development, and virulence in several parasitic protozoa. Here, we discovered increased transcription of the Hsp90 gene under conditions for bradyzoite differentiation, i.e. alkaline and heat shock conditions in vitro, suggesting that Hsp90 may be connected with bradyzoite development in T. gondii. A knockout of the TgHsp90 strain (ΔHsp90) and a complementation strain were constructed. The TgHsp90 knockout cells were found to be defective in host-cell invasion, were not able to proliferate in vitro in Vero cells, and did not show long-time survival in mice in vivo. These inabilities of the knockout parasites were restored upon complementation of TgHsp90. These data unequivocally show that TgHsp90 contributes to bradyzoite development, and to invasion and replication of T. gondii in host cells.

Cryptosporidium is one of the most important parasites in poultry, and this pathogen can infect more than 30 avian species. The present study investigated the infection rate of Cryptosporidium among broiler chicken flocks. A total of 385 fecal samples from broiler chickens in 7 regions of Zhejiang Province collected from November 2010 to January 2012 were examined by microscopy. Thirty-eight (10%) samples were positive for Cryptosporidium infection, and 3 genotypes (Cryptosporidium baileyi, Cryptosporidium meleagridis, and avian genotype II) were identified by PCR and sequencing. A phylogenetic tree of the isolates was analyzed. These results suggest that cryptosporidiosis is widespread in poultry in Zhejiang Province, and is a potential threat to public health as well as the economy. This is the first report about the infection rate and molecular characterization of Cryptosporidium in broiler chickens in Zhejiang.

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]

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]

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]

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]

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]

The first case of human babesiosis was reported in the literature in 1957. The clinical disease has sporadically occurred as rare case reports in North America and Europe in the subsequent decades. Since the new millennium, especially in the last decade, many more cases have apparently appeared not only in these regions but also in Asia, South America, and Africa. More than 20,000 cases of human babesiosis have been reported in North America alone. In several cross-sectional surveys, exposure to Babesia spp. has been demonstrated within urban and rural human populations with clinical babesiosis reported in both immunocompromised and immunocompetent humans. This review serves to highlight the widespread distribution of these tick-borne pathogens in humans, their tick vectors in readily accessible environments such as parks and recreational areas, and their phylogenetic relationships. [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]

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]

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]

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: 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]

• 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]