RESUMEN
Medicinal plants attract the attention of many researchers to find natural and safe remedies for various resistant diseases. Leaves of Mitragyna inermis are widely used in traditional veterinary medicine for the treatment of gastrointestinal strongyles of small ruminants. The aim of the current study is to estimate the antioxidant, anthelmintic and the larval toxicity of the aqueous and hydroethanolic extracts of this plant in addition to the hexane, dichloromethane and ethanol fractions of the hydroethanolic extract. Investigation of the most active extract using Ultra Performance Liquid Chromatography coupled with Quadrupole Time-of-Flight Electrospray Ionization Mass Spectrometry (UPLC-QToF-ESI-MS). Both plant extracts showed good antioxidant activity by scavenging the 2,2'-diphényl-1-picrylhydrazyl (DPPH) radical and reducing the ferric ion. Similarly, they were no-toxic to Artemia salina larvae (CL50 > 0.1 µg/mL). Also, they significantly reduced larval migration and motility of Haemonchus contortus adult worms (p < 0.001). The hexane, dichloromethane and ethanolic fractions of the hydroethanolic extract showed low activity compared to crude extracts except for the hexane fraction on H. contortus adult worms (p < 0.001) while it showed a poor result on larvae. It thus appears that the anthelmintic activity of the extract may be linked to the synergistic action of these compounds. The UPLC-QToF-ESI-MS analysis revealed the tentative identification of 15 compounds including 7 alkaloids. The results of the present study confirm the anthelmintic activity of M. inermis in traditional veterinary medicine.
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Haemonchus contortus is the most prevalent and pathogenic gastrointestinal nematodes (GINs) in ruminants causing extensive economic losses. It is essential to estimate the efficacy of common commercially available anthelmintics against Haemonchus contortus parasite. Here, we standardized an ex-vivo culture platform for H. contortus and evaluated the efficacy of commonly used anthelmintics namely, albendazole (ABZ), levamisole (LVM), ivermectin (IVM), closantel (CLS) and rafoxanide (RFX). Adult worms were collected from abomasa of slaughtered animals, cultured in MEM, DMEM, M199 or RPMI with or without 20% FBS for up to 72 h. Cultured worms were incubated with ABZ, LVM, IVM, RFX or CLS in DMEM supplemented with 20% FBS at different concentrations (0.5-50 µg/ml) in triplicates and examined at 0, 3, 6, 12, 24, 36 and 48 h post treatment. Of the culture conditions, DMEM supplemented with 20% FBS supported the survival of H. contortus for (P < 0.001) longer period of time which was used in the evaluation of anthelmintics. The efficacy of CLS and RFX were significantly (P < 0.001) higher than other drugs and 100% mortality was observed at 2 µg/ml of CLS and RFX within 12 h post treatment. However, ABZ, LVM, and IVM showed significant effect at the concentration of 50 µg/ml with 48, 36, and 24 h, respectively. Morphological changes included severe cuticle disruption around the buccal cavity, posterior region and vulva as well as loss of cuticle structure integrity coupled with expulsion and fragmentation of digestive components of parasites when treated with 50 µg/ml of ABZ, LVM, and IVM and 2 µg/ml of RFX and CLS. Collectively, DMEM supplemented with 20% FBS can be used as ex-vivo culture platform for maintenance of H. contortus, and RFX and CLS can be used as the promising drugs for the prevention, control and treatment of H. contortus infections.
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Haemonchus contortus is an important parasite that causes disease that seriously endangers ruminant animals cattle, sheep, goat, and camel. Here, we compared the proeomics analysis of three adult Haemonchus contortus isolates from mouflons (Ovis ammon). A total of 1,299 adult worm proteins were identified, and 461 proteins were quantified, of which 82 (108), 83 (97), and 97 (86) significantly upregulated (downregulated) differentially expressed proteins (DEPs) were detected among pairwise comparisons (1-vs.-3, 2-vs.-3, and 2-vs.-1). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and bioinformatic analysis indicated that these DEPs are mainly concentrated in cellular composition, molecular function, biological function, and catabolism pathways. In addition, Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were carried out to screen the DEPs. The main biological processes involved were nucleotide, nucleotide phosphate, ribonucleotide, purine-containing compound, purine ribonucleotide, single-organism, oxoacid, organic, carboxylic, oxoacid metabolic processes and single-organism catabolic processes. The majority of KEGG pathways were found to be related to metabolic pathways, biosynthesis of secondary metabolites, biosynthesis of antibiotics, carbon metabolism, and microbial metabolism in diverse environments. Moreover, we also found differences in the expression of some important or novel regulatory proteases, such as serine hydroxymethyl transferase (SHMT), dihydrolipoyl dehydrogenase (DLD), and transket pyr domain-containing protein (TKPD). In summary, label-free proteomic analysis of adult H. contortus worms displayed significant differences in three different individual isolates, which helps to improve our understanding of the growth and metabolic mechanisms of H. contortus in different individuals and relative natural environments and provides novel drug targets for the treatment of parasitic diseases.
Asunto(s)
Haemonchus , Proteómica , Bovinos , Ovinos , Animales , Haemonchus/genética , Haemonchus/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Proteínas del Helminto/genética , Proteínas del Helminto/metabolismo , Cabras/metabolismoRESUMEN
Haemonchosis is a parasitic disease of small ruminants that adversely affects livestock production. Haemonchus contortus is one of the most prevalent nematode parasites that infect the abomasum of small ruminants. This parasite reduces milk production, overall growth and sometimes causes the death of the infected animals. The evaluation of the biocontrol potential of some abomasum bacterial isolates against H. contortus is investigated in this study. Out of which, three isolates-Comamonas testosteroni, Comamonas jiangduensis, Pseudomonas weihenstephanesis-show significant effect against the nematode L3, adult, and egg hatch inhibition assays. Various concentrations of metabolites from these bacteria are prepared and applied in different treatments compared with control. In the case of adult mortality assay, 50% metabolites of C. testosteroni and P. weihenstephanesis show 46% adult mortality, whereas C. jiangduensis shows 40% mortality. It is observed that decreasing the concentration of bacterial metabolite, lowers nematode mortality. The minimum nematode mortality rate is recorded at the lowest filtrates concentration of all the bacterial isolates. The same trend is observed in egg hatch inhibition assay, where the higher concentration of bacterial culture filtrates shows 100% inhibition of H. contortus egg. It is concluded that the effect of bacterial culture filtrates against H. contortus is dose-dependent for their activity against nematode L3, adult, and inhibition of egg hatchment.
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The prevention, treatment and control of Haemonchus contortus have been increasingly problematic due to its widespread occurrence and anthelmintic resistance. There are very few descriptions of recombinant antigens being protective for H. contortus, despite the success of various native antigen preparations, including Barbervax. We recently identified an H. contortus excretorysecretory antigen, H. contortus adhesion-regulating molecule 1 (HcADRM1), that served as an immunomodulator to impair host T-cell functions. Given the prophylactic potential of HcADRM1 protein as a vaccine candidate, we hereby assessed the efficacies of HcADRM1 preparations against H. contortus infection. Parasitological and immunological parameters were evaluated throughout all time points of the trials, including fecal egg counts (FEC), abomasal worm burdens, complete blood counts, cytokine production profiles and antibody responses. Active vaccination with recombinant HcADRM1 (rHcADRM1) protein induced protective immunity in inoculated goats, resulting in reductions of 48.9 and 58.6% in cumulative FEC and worm burdens. Simultaneously, passive administration of anti-HcADRM1 antibodies generated encouraging levels of protection with 46.7 and 56.2% reductions in cumulative FEC and worm burdens in challenged goats. In addition, HcADRM1 preparations-immunized goats showed significant differences in mucosal and serum antigen-specific immunoglobulin G (IgG) levels, total mucosal IgA levels, haemoglobin values and circulating interferon-γ, interleukin (IL)-4 and IL-17A production compared to control goats in both trials. The preliminary data of these laboratory trials validated the immunoprophylactic effects of rHcADRM1 protein. It can be pursued as a potential vaccine antigen to develop an effective recombinant subunit vaccine against H. contortus under field conditions.
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Enfermedades de las Cabras , Hemoncosis , Haemonchus , Animales , Anticuerpos Antihelmínticos , Cabras , Hemoncosis/prevención & control , Hemoncosis/veterinariaRESUMEN
Unlike the successful immunization of native H. contortus antigens that contributed to the realization of the first commercial vaccine Barbervax, not many studies revealed the encouraging protective efficacies of recombinant H. contortus antigens in laboratory trials or under field conditions. In our preliminary study, H. contortus α/ß-hydrolase domain protein (HcABHD) was demonstrated to be an immunomodulatory excretory-secretory (ES) protein that interacts with goat T cells. We herein evaluated the protective capacities of two HcABHD preparations, recombinant HcABHD (rHcABHD) antigen and anti-rHcABHD IgG, against H. contortus challenge via active and passive immunization trials, respectively. Parasitological parameter, antibody responses, hematological pathology and cytokine profiling in unchallenged and challenged goats were monitored and determined throughout both trials. Subcutaneous administration of rHcABHD with Freund adjuvants elicited protective immune responses in challenged goats, diminishing cumulative fecal egg counts (FEC) and total worm burden by 54.0% and 74.2%, respectively, whereas passive immunization with anti-rHcABHD IgG conferred substantial protection to challenged goats by generating a 51.5% reduction of cumulative FEC and a 73.8% reduction of total worm burden. Additionally, comparable changes of mucosal IgA levels, circulating IgG levels, hemoglobin levels, and serum interleukin (IL)-4 and IL-17A levels were observed in rHcABHD protein/anti-rHcABHD IgG immunized goats in both trials. Taken together, the recombinant version of HcABHD might have further application under field conditions in protecting goats against H. contortus infection, and the integrated immunological pipeline of ES antigen identification, screening and characterization may provide new clues for further development of recombinant subunit vaccines to control H. contortus.
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Enfermedades de las Cabras/parasitología , Hemoncosis/veterinaria , Haemonchus/inmunología , Proteínas del Helminto/uso terapéutico , Vacunas/uso terapéutico , Animales , Antígenos Helmínticos/uso terapéutico , Ensayo de Inmunoadsorción Enzimática , Femenino , Enfermedades de las Cabras/prevención & control , Cabras , Hemoncosis/prevención & control , Masculino , Recuento de Huevos de Parásitos/veterinaria , Vacunas Sintéticas/uso terapéuticoRESUMEN
BACKGROUND: Gastrointestinal nematodes could release excretory-secretory (ES) proteins into the host environment to ensure their survival. These ES proteins act as immunomodulators to suppress or subvert the host immune response via the impairment of immune cell functions, especially in chronic infections. In our preliminary study, Haemonchus contortus adhesion-regulating molecule 1 (HcADRM1) was identified from H. contortus ES proteins (HcESPs) that interacted with host T cells via liquid chromatography-tandem mass spectrometry analysis. However, little is known about HcADRM1 as an ES protein which may play a pivotal role at the parasite-host interface. METHODS: Based on bioinformatics approaches, multiple amino acid sequence alignment was conducted and the evolutionary relationship of HcADRM1 with ADRM1 orthologues was extrapolated. Employing RT-qPCR and immunohistochemistry assays, temporal transcriptional and spatial expression profiles of HcADRM1 were investigated. Using immunostaining approaches integrated with immunological bioassays, the immunomodulatory potentials of HcADRM1 on goat T cells were assessed. RESULTS: We hereby demonstrated that HcADRM1 with immunodiagnostic utility was a mammalian ADRM1 orthologue abundantly expressed at all developmental stages of H. contortus. Given the implications of ADRM1 proteins in cell growth, survival and development, we further investigated the immunomodulatory property of HcADRM1 as an individual ES protein acting at the parasite-host interface. The rHcADRM1 stimuli notably suppressed T cell viability, promoted intrinsic and extrinsic T cell apoptosis, inhibited T cell proliferation and induced cell cycle arrest at G1 phase. Simultaneously, rHcADRM1 stimuli exerted critical controls on T cell cytokine secretion profiles, predominantly by restraining the secretions of interleukin (IL)-4, IL-10 and interferon-gamma. CONCLUSIONS: Importantly, HcADRM1 protein may have prophylactic potential for anti-H. contortus vaccine development. Together, these findings may contribute to the clarification of molecular and immunomodulatory traits of ES proteins, as well as improvement of our understanding of parasite immune evasion mechanism in H. contortus-host biology.
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Haemonchus/inmunología , Proteínas del Helminto/inmunología , Inmunomodulación , Animales , Antígenos Helmínticos , Apoptosis , Proliferación Celular , Citocinas/metabolismo , Hemoncosis/inmunología , Hemoncosis/parasitología , Haemonchus/metabolismo , Proteínas del Helminto/metabolismo , Interacciones Huésped-Parásitos , Evasión Inmune , Inmunidad/inmunología , Interferón gamma/metabolismo , Óxido Nítrico/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , VacunasRESUMEN
The α/ß-hydrolase domain (ABHD) proteins belonging to α/ß-hydrolase (ABH) superfamily are ubiquitously distributed throughout all the organisms, and their functional roles have been implicated in energy metabolism, cell signaling, growth and development. In our preliminary work, we identified a novel ABHD protein derived from Haemonchus contortus excretory-secretory (ES) proteins (HcESPs) that interacted with host T cells. Here, we demonstrated that H. contortus ABHD (HcABHD) protein, expressed in all life-cycle stages of H. contortus, is a mammalian ABHD17 homolog with immunodiagnostic utility and lipase activity. Given its catalytic activities and immunomodulatory potentials, we further investigated the functional diversity of HcABHD as an individual ES protein in parasite-host interactions. HcABHD protein may serve as depalmitoylase or thioesterase to suppress cell viability, inhibit cell proliferation, induce intrinsic and extrinsic T cell apoptosis, and cause cell cycle arrested at G1 phase. Moreover, recombinant HcABHD stimuli exerted critical controls on T cell cytokine production profiles, predominantly by inhibiting the secretions of interleukin (IL)-4, interferon-gamma (IFN-γ) and transforming growth factor-beta (TGF-ß) 1, and promoting IL-10 production. As the immunomodulator acting at the parasite-host interface, HcABHD protein may have potential applications for the vaccine development of therapeutic intervention. Together, these findings may help illuminate the molecular and particularly immunomodulatory aspects of ES proteins and contribute to an enhanced understanding of parasite immune evasion in H. contortus-host biology.
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Hemoncosis/inmunología , Haemonchus/inmunología , Proteínas del Helminto/inmunología , Interacciones Huésped-Parásitos/inmunología , Hidrolasas/inmunología , Animales , Femenino , Cabras , Ratas , Ratas Sprague-DawleyRESUMEN
The objective of this study was to analyze the mRNA transcription levels of ten functional genes of P-glycoproteins (P-gp) in free life stages, eggs and infective larvae (L3) and in endoparasitic stages, fourth larval stage (L4) and adult males of two native isolates of Haemonchus contortus: resistant and susceptible to IVM. The IVM resistant isolate was obtained from sheep naturally infected with H. contortus, and the susceptible isolate (with no pressure to IVM) conserved since 1990. The lethal effect of IVM was evaluated under in vitro conditions, which showed significant differences between susceptible and resistant H. contortus L3 isolates (P < 0.01). The IVM susceptible isolate revealed a lethal effect of 79.22% at 11.42 mM, whereas that resistant isolate showed no lethal effect at any of the four assessed concentrations (1.43, 2.85, 5.71 and 11.42 mM) of IVM. The expression levels of ten Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14, and 16) were evaluated in the resistant isolate of H. contortus and compared to the susceptible isolate (as control), using two constitutive genes (GAPDH and ß-tubulin). Up-regulation at two statistical significant values (P ≤ 0.05, 0.1) was the criterion to associate IVM resistance with the free life and endoparasitic stages of H. contortus. The expression levels in H. contortus adult nematodes showed 5.64 to 127.56-fold increase for Hco-pgp genes 1, 9, 12, 14, and 16, followed by an increase for Hco-pgp-2 (49.75-fold) and Hco-pgp-10 (106.40-fold) in L4, and for Hco-pgp-16 (2.90-fold) in eggs (P ≤ 0.05). In addition, high expression levels with P < 0.1 were detected in H. contortus L3, L4, and adults for Hco-pgp genes 1, 4, 11, 12, and 16, with changes ranging from 2.17 to 29.72-fold. In conclusion, the highest expression was observed in the adult stage of H. contortus, and the most frequent gene with a significant P-value was Hco-pgp-16, revealing it plays an important role in IVM resistance.
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Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Antihelmínticos/farmacología , Resistencia a Medicamentos/genética , Haemonchus/efectos de los fármacos , Proteínas del Helminto/genética , Ivermectina/farmacología , ARN Mensajero/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Expresión Génica , Hemoncosis , Haemonchus/genética , Haemonchus/metabolismo , Proteínas del Helminto/metabolismo , Larva/efectos de los fármacos , Larva/genética , Larva/metabolismo , Masculino , Pruebas de Sensibilidad Parasitaria , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ovinos , Regulación hacia Arriba , Cigoto/citología , Cigoto/efectos de los fármacos , Cigoto/metabolismoRESUMEN
Bridelia ferruginea (Euphorbiaceae) and Mitragyna inermis (Rubiaceae) are two plants of the beninese pharmacopeia used in vivo for the control of gastrointestinal nematodes (GINs) in small ruminants. The objective of the present study is to explore the mechanism of bioactive compounds involved in the action of these two plants on the third-stage infective larvae (L3s) of Haemonchus contortus. Thus, sheathed L3s of H. contortus were incubated with acetone extracts of B. ferruginea and M. inermis at concentrations of 0, 150, 300, 600 and 1200 µg/mL for 3 h at 25°C. The L3s were then washed and artificially submitted to exsheathment in the presence of sodium hypochlorite solution. The role of tannins was verified by adding a tannin inhibitor, polyvinyl polypyrrolidone (PVPP), to the acetone extracts of these two plants for 2 h at 25°C. Acetone extracts from B. ferruginea and M. inermis inhibited the exsheathment of H. contortus larvae (p < 0.001) and this inhibitory effect was dose-dependent for M. inermis at the concentrations tested. Treatment of B. ferruginea and M. inermis extracts with PVPP was associated with a partial restoration of the exsheathment kinetics of H. contortus larvae (p < 0.001), confirming the predominant role of tannins but also the residual role of other secondary metabolites. These in vitro results suggest that these plants are endowed with anthelmintic (AHs) properties and therefore likely to be used as alternatives to synthetic molecules.
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OBJECTIVE: This study aimed to explore the phytochemical constituents and anthelmintic activities of four Cassia spp. leaves against Haemonchus contortus. MATERIALS AND METHODS: The extracts were prepared from four species of Cassia spp. (C. siamea, C. fistula, C. surattensis, and C. spectabilis). Phytochemical screening of the extract was done based on the Harborne method. Evaluation of the anthelmintic activities against H. contortus was done in vitro using infective larvae (L3) migration inhibition assay (LMIA). Measurement of larvae migrating was conducted through a nylon filter with a pore size of 20 µm. The doses of Cassia spp. extract implemented were 25, 50, 100, and 200 mg/ml. RESULTS: Tannins, alkaloids, phenol hydroquinone, flavonoids, steroids, triterpenoids, and saponins were present in all the extracts, whereas alkaloids were absent in C. fistula. No triterpenoids were found in C. surattensis and C. spectabilis. Movement of H. contortus larvae was significantly inhibited after exposure to Cassia extracts at various dosage levels (p < 0.05). The test results using LMIA on L3 H. contortus showed the lowest inhibition in the negative control. Among the species of Cassia, the C. surattensis (at 200 mg/ml) showed the highest (p < 0.05) inhibition level on the larvae. The latter result corresponded to the effect of albendazole. CONCLUSION: Compared to other Cassia spp., C. surattensis exhibited the highest inhibition against L3 H. contortus. However, the inhibition effect of C. surattensis was still lower as compared to albendazole.
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Benzimidazoles have been intensively (for over 40 years) used in the livestock sector, particularly in small ruminants. This has been led to the widespread emergence of resistance in a number of small ruminant parasite species, especially Haemonchus contortus. In many countries benzimidazole resistance has severely compromised the control of H. contortus in small ruminants; but there is a little information on benzimidazole resistance in H. contortus infecting buffalo and cattle. Resistance to benzimidazoles have also been reported in the large ruminant parasite, Haemonchus placei, but again there is relatively little information on its prevalence. Hence it is very important to understand how resistance-conferring mutations emerge and spread in both parasites in buffalo and cattle hosts in order to develop approaches for the recognition of the problem at an early stage of its development. The present study suggests that the F200Y (TAC) mutation is common in H. contortus, being detected in 5/7 populations at frequencies between 7 and 57%. Furthermore, 6/10 H. placei populations contained the F200Y (TAC) mutation, albeit at low frequencies of between 0.4 and 5%. The phylogenetic analysis suggests that the F200Y (TAC) mutation in H. contortus has emerged on multiple occasions in the region, with at least three independent emergences across the populations. In contrast, the F200Y (TAC) resistance-conferring mutation in H. placei is only seen on a single haplotype. A high level frequency of the resistance haplotypes in the region, suggests that the unique resistance conferring-mutation has spread from a single emergence; likely by anthropogenic animal movement. Overall, these results provide the first clear genetic evidence for the spread of benzimidazole resistance-conferring mutations to multiple different locations from a single emergence in H. placei; while being consistent with previous small ruminant-based observations of multiple emergence of resistance mutations in H. contortus.
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Antihelmínticos/farmacología , Bencimidazoles/farmacología , Búfalos/parasitología , Enfermedades de los Bovinos/parasitología , Hemoncosis/veterinaria , Haemonchus/efectos de los fármacos , Animales , Antihelmínticos/uso terapéutico , Bencimidazoles/uso terapéutico , Bovinos , Enfermedades de los Bovinos/tratamiento farmacológico , Resistencia a Medicamentos/genética , Hemoncosis/tratamiento farmacológico , Hemoncosis/parasitología , Haemonchus/genética , Mutación , FilogeniaRESUMEN
Drug resistance in helminth parasites has incurred several difficulties to livestock industry and ranked among the top public health concerns. Therefore, seeking for new agents to control parasites is an urgent strategy. In the recent years, metallic nanoparticles have been considerably evaluated for anthelmintic effects. The current research was conducted to assess possible anthelmintic impacts of zinc oxide nanoparticles (ZnO-NPs) on a prevalent gastrointestinal nematode, H. contortus. Moreover, several biomarkers of oxidative/nitrosative stress and DNA damage were measured. Various concentrations of the nanoparticle were prepared and incubated with the worms for 24 hours. The parasite mobility, mortality rate, antioxidant enzymes activities (SOD, Catalase and GSH-Px), lipid peroxidation, total antioxidant status as well as nitric oxide (NO) contents and DNA damage were determined. ZnO-NPs exerted significant wormicidal effects via induction of oxidative/nitrosative stress and DNA damage. Conclusively, ZnO-NPs can be utilized as a novel and potential agent to control and treatment of helminth parasitic infections.
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Antihelmínticos/farmacología , Biomarcadores/análisis , Hemoncosis/veterinaria , Haemonchus/efectos de los fármacos , Nanopartículas del Metal/química , Óxido de Zinc/farmacología , Animales , Antioxidantes/metabolismo , Daño del ADN/efectos de los fármacos , Femenino , Hemoncosis/parasitología , Hemoncosis/prevención & control , Haemonchus/genética , Haemonchus/fisiología , Peroxidación de Lípido/efectos de los fármacos , Ganado , Óxido Nítrico/análisis , Estrés Nitrosativo/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Faecal worm egg counts (FEC) are very important indicators in assessing the intensity of helminth infections in animal hosts and inform decisions taken in parasite control programmes. FEC are however affected by many factors which include the dose level of larval challenge, species composition of the worms, worm burden, female worm fecundity and concurrent infections to name but these few. The relevance of time of collection of faecal sample for FEC is not fully known and in most cases faeces for analysis is collected whenever feasible for the researcher on sample collection days. The significance of the time of collection of faeces on FEC was therefore investigated at two different periods of the day, morning and afternoon. Faecal samples were collected at 7-8 a.m. (morning) and at 2-3 p.m. (afternoon) on same sampling days from 6 mice and 10 West African Dwarf goat kids experimentally infected with Heligmosomoides bakeri and Haemonchus contortus respectively. FEC were conducted using the flotation and modified McMaster techniques. Overall, the 2-3 p.m. FEC tended to be higher than the 7-8 a.m. FEC in both animal species but the difference was not significant (P > 0.05). The time of collection of faecal samples for faecal worm egg counts is therefore not a crucial factor that may significantly affect FEC in H. bakeri and H. contortus infected mice and goats respectively although it would be advisable to maintain particular periods for collection of faeces in particular experiments for best results of FEC to be obtained.
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In vitro studies using plant extracts suggest a relationship between their polyphenol contents and their anthelmintic (AH) activity against Haemonchus contortus. High polyphenol content appears to increase the efficacy of plant extracts against H. contortus as assessed by the larval exsheathment inhibition assay (LEIA) while appearing to reduce the AH efficacy measured using the egg hatch assay (EHA). In addition, some plants lack AH activity. Therefore, the present study investigated the relationship between the contents of condensed tannins (CT), total phenols (TP), and total tannins (TT) in methanol:water extracts (70:30) obtained from ten tropical plant species consumed by small ruminants as well as their AH activity against H. contortus evaluated by LEIA and EHA. Extracts of Acacia collinsii, Lysiloma latisiliquum, Havardia albicans, Senegalia gaumeri, Mimosa bahamensis, Piscidia piscipula, Acacia pennatula, Gymnopodium floribundum, Leucaena leucocephala, and Bunchosia swartziana were examined. Positive correlations were found between the effective concentration 50% (EC50) (EHA) of extracts and their CT (r = 0.6809, P < 0.05, n = 10) and TP (r = 0.9152, P < 0.05, n = 10) content, suggesting that their concentration negatively affected AH activity against eggs. Based on the LEIA, there was no significant association between the EC50 and the CT, TP, or TT of all extracts evaluated. Thus, if sheep and goats consume a complex feed mixture with high amounts of CT, TP, and TT, it might be difficult to observe an AH effect against H. contortus egg hatching. However, the AH effect upon L3 establishment might be feasible.
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Antihelmínticos/farmacología , Haemonchus/efectos de los fármacos , Extractos Vegetales/farmacología , Animales , Antihelmínticos/química , Larva/efectos de los fármacos , Fenoles/farmacología , Extractos Vegetales/química , Ovinos , Taninos/análisis , Taninos/farmacologíaRESUMEN
Microtubules are non-covalent cylindrical polymers formed by alpha- and beta-tubulin heterodimer units, crucial for cell division, intracellular transport, motility and differentiation. This makes them very attractive pharmacological targets exploited to develop different drugs such as anthelmintics, antifungals, and antineoplastics. In this work, in order to establish an in vitro target-based screen to integrate to the search for new anthelmintics, we explored the extraction of native assembly-competent tubulin from two helminth parasites: Mesocestoides vogae tetrathyridia (syn. corti, Cestoda: Cyclophyllidea), a useful cestode biological model, and Haemonchus contortus, a sheep gastrointestinal nematode of interest in livestock production. For this purpose, a novel tubulin affinity chromatography procedure was employed, based on the binding capacity of TOG (Tumor Overexpressed Gene) domain from MAPs (microtubule-associated proteins). The TOG domain of the protein Stu2 from Saccharomyces cerevisiae fused to GST (glutathione S- transferase) were produced in E. coli, and the immobilized recombinant proteins allowed for native tubulin extraction from parasites. The binding capacity of TOG1 affinity column (3.6%) was estimated using commercial porcine brain tubulin. A total amount of up to 126 µg of M. vogae tubulin was purified, whereas H. contortus tubulin co-eluted with glutamate dehydrogenase enzyme. The identity of tubulins was confirmed by western blotting and mass spectrometry. The abundance of tubulin estimated in M. vogae was 10% soluble extract, which probably could explain differences observed between tubulin purification results of both helminth parasites.
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Cromatografía de Afinidad/métodos , Haemonchus/química , Mesocestoides/química , Proteínas Asociadas a Microtúbulos/metabolismo , Tubulina (Proteína)/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Infecciones por Cestodos/parasitología , Escherichia coli/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Hemoncosis/parasitología , Hemoncosis/veterinaria , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ovinos , Enfermedades de las Ovejas/parasitología , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Porcinos , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMEN
Investigations into the pharmacology of different types of cys-loop GABA receptor have relied for years on the chemical modification of GABA-like compounds. The GABA metabolite GABOB is an attractive molecule to modify due to its convenient chemical structure. In the process of developing new GABA-mimic compounds from GABOB as a starting compound three small molecule GABA derivatives were synthesized using a variety of chemical transformations. Amongst these, a new and reliable method to synthesize TACA (trans-4-aminocrotonic acid) is reported.
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Crotonatos/farmacología , Agonistas de Receptores de GABA-A/farmacología , Receptores de GABA-A/metabolismo , Animales , Crotonatos/síntesis química , Crotonatos/química , Relación Dosis-Respuesta a Droga , Agonistas de Receptores de GABA-A/síntesis química , Agonistas de Receptores de GABA-A/química , Estructura Molecular , Oocitos/efectos de los fármacos , Relación Estructura-Actividad , Xenopus laevisRESUMEN
We conducted herein transcriptome sequencing of the ovine abomasal tissues using the Illumina HiSeq 4000 platform to segregate early and late H. contortus-infected sheep (7 and 50days post-infected groups, respectively) from the control naive ones. A total of 548, 357 and 7 were substantially induced genes in 7days post-infection versus uninfected-control group, 50days post-infection versus 7days post-infection (7dpi), and 50days post-infection (50dpi) versus uninfected-control group, respectively. However, a total of 301, 355 and 11 were significantly repressed genes between 7dpi versus uninfected-control group, 50dpi versus 7dpi, and 50dpi versus uninfected-control group, correspondingly. This indicates that H. contortus infection induced a more potent activation of abomasal gene expression in the early stage of infection as compared to the late stage. Seven pathways were annotated by Kyoto Encyclopedia of Genes, and Genomes pathway analysis accounted for the significant percentage in early H. contortus infection. This study shows for the first time that both galectin-11 and matricellular protein osteopontin are up-regulated in abomasal tissue after chronic H. contortus infection, while galectin-4 is specifically down-regulated in the early infection. Additionally, our results showed that the induction or repression of these molecules is likely to determine the infection progression.
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Abomaso/parasitología , Mucosa Gástrica/parasitología , Perfilación de la Expresión Génica/métodos , Hemoncosis/veterinaria , Haemonchus/fisiología , Abomaso/metabolismo , Animales , Regulación hacia Abajo , Galectinas/genética , Mucosa Gástrica/metabolismo , Ontología de Genes , Hemoncosis/genética , Hemoncosis/parasitología , Osteopontina/genética , Ovinos , Transcriptoma , Regulación hacia ArribaRESUMEN
To evaluate the role of IL-13 gene in Garole/Sahabadi sheep against resistant/susceptibility to Haemonchus contortus five Garole sheep (Group I) with consistently showing low egg per gram of faeces (EPG) (≤150) and another five Garole sheep (Group II) with high EPG (≥500) were selected and fed orally with active H. contortus third stage (L3) larvae at the dose rate of 500 larvae/kg body weight. Five Sahabadi sheep (Gr-III) known susceptible to H. contortus were also challenged with the same dose as the previous groups. Blood was collected from each group on day 0, 7 and 14days post infection (dpi). The serum level of IL-13 cytokine was evaluated by ELISA at different days of post infection. The ovine IL-13 was partially isolated and the RNAm level of IL-13 expression was studied by real-time PCR at different days post infection. In the resistant Garole sheep (Gr-I) the concentration of IL-13 cytokine increased significantly (P≤0.05) 14days PI compared to susceptible Garole (Gr-II)/Sahabadi (Group-III). In similar way the relative expression of IL-13 in group I (Gr-I) was found more than seven fold higher than Gr-II and Gr-III on 14 dpi. The Th2 type immune response to H. contortus characterized by higher serum level of IL-13 cytokine and IL-13 gene expression indicated that IL-13 gene plays a vital role for the reduced establishment and/or survival of established H. contortus in resistant Garole sheep.
RESUMEN
ADP-ribosylation factors (ARFs) are members of the Ras-related small GTPase family involved in the vesicular trafficking regulation. Immunomodulatory effects of these proteinson host cell arenot being addressed yet. H. contortus small GTPase ADP-ribosylation 1 gene (HcARF1) was cloned and recombinant protein of HcARF1 (rHcARF1) was successfully expressed in Escherichia coli. Binding activity of rHcARF1 to goat PBMCs was confirmed by immunofluorescence assay (IFA) and its immunomudulatory effects on cytokine secretion, cell proliferation, cell migration and nitric oxide production (NO) were observed by co-incubation of rHcARF1. IFA results revealed that rHcARF1 could bind to the PBMCs. The interaction of rHcARF1 modulated the cytokine production, the production of IL-4, IL-10 and IL-17 was increased in a dose dependent manner, however, the IFN-γ production was significantly decreased. Cell migration and NO production were significantly increased by rHcARF1, whereas, rHcARF1 treatment significantly suppressed the proliferation of the PBMC in a dose dependent manner. Our findings showed that the rHcARF1 play important roles on the goat PBMCs.