RESUMO
The objective of this study was to identify genomic regions and genes associated with resistance to gastrointestinal nematodes in Australian Merino sheep in Uruguay, using the single-step GWAS methodology (ssGWAS), which is based on genomic estimated breeding values (GEBVs) obtained from a combination of pedigree, genomic, and phenotypic data. This methodology converts GEBVs into SNP effects. The analysis included 26,638 animals with fecal egg count (FEC) records obtained in two independent parasitic cycles (FEC1 and FEC2) and 1700 50K SNP genotypes. The comparison of genomic regions was based on genetic variances (gVar(%)) explained by non-overlapping regions of 20 SNPs. For FEC1 and FEC2, 18 and 22 genomic windows exceeded the significance threshold (gVar(%) ≥ 0.22%), respectively. The genomic regions with strong associations with FEC1 were located on chromosomes OAR 2, 6, 11, 21, and 25, and for FEC2 on OAR 5, 6, and 11. The proportion of genetic variance attributed to the top windows was 0.83% and 1.9% for FEC1 and FEC2, respectively. The 33 candidate genes shared between the two traits were subjected to enrichment analysis, revealing a marked enrichment in biological processes related to immune system functions. These results contribute to the understanding of the genetics underlying gastrointestinal parasite resistance and its implications for other productive and welfare traits in animal breeding programs.
Assuntos
Polimorfismo de Nucleotídeo Único , Doenças dos Ovinos , Animais , Ovinos/parasitologia , Ovinos/genética , Doenças dos Ovinos/genética , Doenças dos Ovinos/parasitologia , Resistência à Doença/genética , Estudo de Associação Genômica Ampla , Infecções por Nematoides/genética , Infecções por Nematoides/veterinária , Infecções por Nematoides/parasitologia , Austrália , Contagem de Ovos de Parasitas/veterinária , Enteropatias Parasitárias/genética , Enteropatias Parasitárias/veterinária , Enteropatias Parasitárias/parasitologiaRESUMO
This study assessed the immunoprotective effect in lambs of a native excreted/secreted 15-kDa protein and two synthesised S28 peptides derived from the infective transitory larvae (xL3) and adult stages (AS) of Haemonchus contortus. Twenty-two Pelibuey lambs were divided into negative and positive control groups, as well as immunised lamb groups, with 100 µg of the 15-kDa native protein (15kDaNP) and S28 peptides (S28P). The eggs per gram (EPG) and haematocrit were measured, and AS were counted and morphologically measured. To assess the immunoprotection in lambs, indirect enzyme-linked immunosorbent assays and relative expression analyses of immune cytokines were performed using serum and abomasal samples. Our results showed a 72.28% reduction in adult worms (AW) in the 15kDaNP-immunised group, achieving a high clinical response with 41% haematocrit and low EPG values (436 ± 661). Conversely, the S28P group achieved the highest IgG levels (2.125 ± 0.880 OD), with AW exhibiting the greatest body length (p > 0.05) and upregulation of the IL5 and FCεR1A genes associated with nematode control. The 15kDaNP group showed increased expression of genes related to nematode control and anti-inflammatory responses, including IL4, IL5, IL6, and IL13 (p < 0.05). The S28P and 15kDaNP should be explored as potential vaccines against sheep haemonchosis.
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The variability in the expression of different P-glycoprotein (P-gp) genes in parasitic nematodes of ruminants such as Haemonchus contortus (Hco-pgp) may be caused by different factors including nematode biology, geographical region and anthelmintic pressure. This study analysed the relative expression level of 10 P-gp genes in two H. contortus (Hco-pgp) field isolates from Yucatan, Mexico: 1) PARAISO (IVM-resistant) and 2) FMVZ-UADY (IVM-susceptible). These isolates were compared with a susceptible reference isolate from Puebla, Mexico, namely "CENID-SAI". In all cases H. contortus adult males were used. The Hco-pgp genes (1, 2, 3, 4, 9, 10, 11, 12, 14 and 16) were analysed for each isolate using the RT-qPCR technique. The Hco-pgp expressions were pairwise compared using the 2-ΔΔCt method and a t-test. The PARAISO isolate showed upregulation compared to the CENID-SAI isolate for Hco-pgp 1, 3, 9, 10 and 16 (P < 0.05), and the PARAISO isolate showed upregulation vs. FMVZ-UADY isolate for Hco-pgp 2 and 9 (P < 0.05), displaying 6.58- and 5.93-fold differences (P < 0.05), respectively. In contrast, similar Hco-pgp gene expression levels were recorded for FMVZ-UADY and CENID-SAI isolates except for Hco-pgp1 (P <0.1), which presented a significant upregulation (6.08-fold). The relative expression of Hco-pgp allowed confirming the IVM-resistant status of the PARAISO isolate and the IVM-susceptible status of the FMVZ-UADY isolate when compared to the CENID-SAI reference isolate. Therefore, understanding the association between the Hco-pgp genes expression of H. contortus and its IVM resistance status could help identifying the genes that could be used as molecular markers in the diagnosis of IVM resistance. However, it is important to consider the geographic origin of the nematode isolate and the deworming history at the farm of origin.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Resistência a Medicamentos , Hemoncose , Haemonchus , Ivermectina , Animais , Masculino , Anti-Helmínticos/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Expressão Gênica , Hemoncose/veterinária , Hemoncose/parasitologia , Haemonchus/efeitos dos fármacos , Haemonchus/genética , Ivermectina/farmacologia , México , Fenótipo , Ovinos , Doenças dos Ovinos/parasitologiaRESUMO
BACKGROUND: Arthrobotrys species are nematophagous fungi that secrete extracellular nematocidal products (ECP). The individual and combined effects of ECP from Arthrobotrys oligospora (Ao) and A. musiformis (Am) growth in liquid media against Haemonchus contortus L3 (HcL3) were assessed. METHODS: The isolation, morphological (MI) and molecular identification (Mol-I), assessment of nematocidal activity (NA) of fungal liquid culture filtrates (LCF) in two liquid media alone and in combination and the myco-compound profile identification (MCP) were performed. RESULTS: The MI suggested that the fungi corresponded to the species Ao and Am. This result was confirmed by PCR analysis followed by sequencing, alignment and a phylogenetic analysis. Likewise, the highest Hc mortalities were 91.4% with individual LCF of Am and 86.2% with those of Ao at the highest concentration (100 mg/mL) in Czapek-Dox Broth. The combination of both LCF resulted in a similarly high larval mortality with no statistical differences in relation to individual activity (p > 0.05). The MCP showed the presence of alkaloids in both fungi. Coumarins, sterols and saponins were found only in Ao. MAIN CONCLUSIONS: Both fungi produced ECP with a high NA that could be identified and assessed in future studies as potential natural anthelmintic compounds.
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Due to the increasing populations of anthelmintic-resistant gastrointestinal nematodes and as a consequence of the adverse effects of synthetic drugs, this study focuses on the search for secondary metabolites with nematocidal activity from the edible mushroom Pleurotus djamor using The proton nuclear magnetic resonance (1H-NMR) metabolomics. The highest activity was shown by the ethyl acetate fractions of mycelium (EC50 290.8 µg/mL) and basidiomes (EC50 282.7 µg/mL). Principal component analysis (PCA) and hierarchical data analysis (HCA) of the 1H-NMR metabolic profiles data showed that the ethanolic extracts, the ethyl acetate, butanol, and water fractions from mycelium have different metabolic profiles than those from basidiomes, while low polarity (hexane) fractions from both stages of fungal development show similar profiles. Orthogonal partial least squares discriminant analysis (OPLS-DA) allowed the identification of signals in the 1H-NMR metabolic profile associated with nematocidal activity. The signals yielded via OPLS-DA and bidimensional NMR analysis allowed the identification of uracil as a component in the ethyl acetate fraction from basidiomes, with an EC50 of 237.7 µg/mL. The results obtained showed that chemometric analyses of the 1H-NMR metabolic profiles represent a viable strategy for the identification of bioactive compounds from samples with complex chemical profiles.
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Biomarkers are specific molecular, histological, or physiological characteristics of normal or pathogenic biological processes and are promising in the diagnosis of gastrointestinal nematodes (GINs). Although some biomarkers have been validated for infection by Ostertagia sp. in cattle raised in temperate regions, there is a lack of information for tropical regions. The aim of this project was to assess potential biomarkers and validate the most promising. In the first study, 36 bovines (Nelore breed) naturally infected by GINs were distributed into two groups: infected (not treated with anthelmintic) and treated (treated with fenbendazole on days 0, 7, 14, 21, 28, 42, and 56). The variables of interest were live weight, fecal egg count, hemogram, serum biochemical markers, phosphorus, gastrin, and pepsinogen. In the second step, pepsinogen was assessed in cattle of the Nelore breed distributed among three groups: infected (not treated with anthelmintic), MOX (treated with moxidectin), and IVM + BZD (treated with ivermectin + albendazole). In the first study, no difference between groups was found for weight, albumin, hematocrit (corpuscular volume [CV]), erythrocytes, or hemoglobin. Negative correlations were found between pepsinogen and both CV and albumin, and albumin was negatively correlated with the percentage of Haemonchus sp. in the fecal culture. Among the biomarkers, only pepsinogen differentiated treated and infected (beginning with the 28th day of the study). In the second study, a reduction in pepsinogen was found after anthelmintic treatment. Therefore, pepsinogen is a promising biomarker of worms in cattle naturally infected by the genera Haemonchus and Cooperia in tropical areas.
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Biomarcadores , Doenças dos Bovinos , Fezes , Infecções por Nematoides , Clima Tropical , Animais , Bovinos , Doenças dos Bovinos/parasitologia , Doenças dos Bovinos/tratamento farmacológico , Biomarcadores/sangue , Infecções por Nematoides/veterinária , Infecções por Nematoides/parasitologia , Infecções por Nematoides/tratamento farmacológico , Fezes/parasitologia , Contagem de Ovos de Parasitas , Anti-Helmínticos/uso terapêutico , Nematoides/isolamento & purificação , Nematoides/classificação , Nematoides/efeitos dos fármacos , Gastroenteropatias/parasitologia , Gastroenteropatias/veterinária , Enteropatias Parasitárias/veterinária , Enteropatias Parasitárias/parasitologia , Fenbendazol/uso terapêuticoRESUMO
This study describes the in vitro anthelmintic effect of a hydroalcoholic extract (HA-E) and its fractions from Cyrtocarpa procera fruits against Haemonchus contortus eggs and infective larvae. The HA-E was subjected to bipartition using ethyl acetate, which resulted in an aqueous fraction (Aq-F) and an organic fraction (EtOAc-F). The HA-E and both fractions were tested using the egg hatching inhibition assay (EHIA) and the larval mortality test (LMT). Fractionation of the EtOAc-F was achieved using different chromatographic processes, i.e., open glass column and HPLC analysis. Fractionation of the EtOAc-F gave 18 subfractions (C1R1-C1R18), and those that showed the highest yields (C1R15, C1R16, C1R17 and C1R18) were subjected to anthelmintic assays. The HA-E and the EtOAc-F displayed 100% egg hatching inhibition at 3 and 1 mg/mL, respectively, whereas Aq-F exhibited 92.57% EHI at 3 mg/mL. All subfractions tested showed ovicidal effect. Regarding the larval mortality test, HA-E and EtOAc-F exhibited a larvicidal effect higher than 50% at 50 and 30 mg/mL, respectively. The subfractions that showed the highest larval mortality against H. contortus were C1R15 and C1R17, with larval mortalities of 53.57% and 60.23% at 10 mg/mL, respectively. Chemical analysis of these bioactive subfractions (C1R15 and C1R17) revealed the presence of gallic acid, protocatechuic acid, and ellagic acid. This study shows evidence about the ovicidal and larvicidal properties of C. procera fruits that could make these plant products to be considered as a natural potential anthelmintic agents for controlling haemonchosis in goats and sheep.
Assuntos
Anti-Helmínticos , Frutas , Haemonchus , Larva , Óvulo , Extratos Vegetais , Animais , Haemonchus/efeitos dos fármacos , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Larva/efeitos dos fármacos , Frutas/química , Anti-Helmínticos/farmacologia , Anti-Helmínticos/isolamento & purificação , Anti-Helmínticos/química , Óvulo/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Ovinos , Hemoncose/parasitologia , Hemoncose/veterinária , Doenças dos Ovinos/parasitologiaRESUMO
Haemonchus contortus and Trichostrongylus colubriformis are the most important gastrointestinal nematodes causing serious losses in sheep production of tropical and subtropical regions. Prophylaxis of gastrointestinal nematode infections is based on anthelmintics use, but their frequent administration selects multiple-resistant parasites. To evaluate how the situation has changed over the last decades, the anthelmintic resistance status of gastrointestinal nematodes in sheep flocks was assessed in the current study and compared to previous surveys. In each one of the 15 flocks evaluated, animals (n ≥ 7) were allocated into at least five groups and treated as follows: 1) untreated control; 2) albendazole; 3) levamisole; 4) ivermectin; and 5) monepantel. If more animals were available, two additional groups were included: 6) closantel, and 7) moxidectin. The faecal egg count reduction test (FECRT) was carried out to evaluate the pre- and post-treatment using the SHINY tool. Haemonchus spp. was the most prevalent nematode from faecal cultures. The mean efficacy of albendazole was 40%. Only in two farms, levamisole presented a relatively high percentage of reduction in the FECRT about 90%, while ivermectin and moxidectin presented the worst mean efficacy of 34% and 21% among all farms, respectively. Like other anthelmintics, closantel demonstrated low efficacy (63%) across all farms evaluated. Monepantel presented an overall mean efficacy of 79%, but it was the only anthelmintic that presented efficacy ≥95%, in five farms. The results revealed that gastrointestinal nematodes with multiple anthelmintic resistance were prevalent in all 15 sheep herds. The research suggests that nematodes are becoming more and more resistant to various anthelmintic compounds, which has made the problem worse. This circumstance highlights the necessity to put into practice sustainable and long-lasting methods to prevent gastrointestinal nematode infections in sheep husbandry.
Assuntos
Aminoacetonitrila/análogos & derivados , Anti-Helmínticos , Haemonchus , Macrolídeos , Nematoides , Infecções por Nematoides , Salicilanilidas , Doenças dos Ovinos , Animais , Ovinos , Levamisol/farmacologia , Levamisol/uso terapêutico , Ivermectina/uso terapêutico , Albendazol/uso terapêutico , Brasil/epidemiologia , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Infecções por Nematoides/tratamento farmacológico , Infecções por Nematoides/epidemiologia , Infecções por Nematoides/veterinária , Fezes/parasitologia , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/epidemiologia , Doenças dos Ovinos/parasitologia , Contagem de Ovos de Parasitas/veterinária , Resistência a MedicamentosRESUMO
Mushrooms of the genus Pleurotus have shown nematophagous activity as it produces many chemical compounds and enzymes affecting parasitic nematodes. This study aimed to extract the inhibitory activity of the five strains of the fungus Pleurotus spp. It was evaluated against eggs and larvae of Haemonchus contortus. The extract of P. ostreatus obtained the highest level of inhibition of eggs at 97.6% (1341 µg/mL) followed by P. pulmonarius (EPP) at 81.2% (774 µg/mL). The extract selected for evaluation against larvae was P. pulmonarius, showing no effect for L3 larvae, but for L4 larvae an immobility effect of 56.93% was observed at 900 µg/mL. The protein profile showed the presence of 23 protein bands in the extract. The crude extract of P. pulmonarius showed degradation of tissues both inside the eggs and larvae L1. Metabolites produced by Pleurotus mushrooms can consider using in agriculture sustainable by utilizing in producing of ovicidal and larvicidal against H. contortus instead of chemical compounds.
Assuntos
Agaricales , Haemonchus , Pleurotus , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/química , LarvaRESUMO
This is the first record of the fungus Flavocillium subprimulinum in Mexico. The isolate was taxonomically characterised and cultured in potato dextrose broth (PDB), Czapek-Dox broth (CzDoxB), and sweet potato dextrose broth (SPDB) to obtain its filtrates (FLCF). The nematocidal activity (NA) of three FLCF concentrations was assessed against Haemonchus contortus L3. Protease activity (PA) was assessed with SDS-PAGE, followed by a zymogram. The NA of the FLCF reached 94.43% in PDB and 95.82% in CzDoxB, respectively, at 100 mg/mL. Lower mortality (64%) was found in SPDB at 100 mg/mL. SDS-PAGE showed bands (in PBS) of ~25, ~40, and ~55 kDa. The zymogram showed protein bands (PBs) with PA in the media, including PBs of ~14, ~40, and ~55 kDa. This study establishes the basis for exploring the potential use of this fungus against H. contortus, which is considered the most pathogenic parasite affecting lambs.
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The current study was designed to check the anthelmintic activities of some local plants. Seeds of Amomum (A.) subulatum and Vitex (V.) negundo in different solvents were subjected to in vitro (adult motility assay; AMA and egg hatch assay; EHA) and in vivo (faecal egg count reduction test; FECRT) anthelmintic activity testing protocols using Haemonchus (H.) contortus as an experimental model. The results of AMA, EHA, and FECRT were statistically analysed through linear regression and Duncan multiple range test. In AMA test, at 50 mg mL-1 concentration, the percent mortality of H. contortus was higher in A. subulatum than V. negundo, whereas, in EHA test, A. subulatum was proven better ovicidal (LC50=14.2 µg mL-1) than V. negundo (LC50= 65.7405 µg mL-1). The FECRT also indicated the better efficacy of A. subulatum than V. negundo against natural infection of gastrointestinal (GI) parasites. The crude powder of plants used in this study showed 29.6% to 57.7% anthelmintic. The reduction rate was found higher for A. subulatum (3 g kg-1) as compared to V. negundo (7 g kg-1). Reagrding efficacy analysis of solvents used for plants extract, ethyl acetate and chloroform were found better in increasing ovicidal activity in adult worms (in vitro testing), whereas, the crude aqueous methanol was found better than the crude powders in in vivo testing. It will be beneficial to document the indigenous knowledge to standard scientific procedures for their validation. This study will help to motivate the farmers to make a better choice of cultivation of the indigenous plants because of their varying efficacies as an alternative preventive approach against the GI parasitic infections.
O presente estudo foi desenhado para verificar as propriedades anti-helmínticas de algumas plantas locais. Sementes de Amomum (A.) subulatum e Vitex (V.) negundo em diferentes solventes foram submetidas à análise de atividade anti-helmíntica in vitro (ensaio de motilidade de adultos; AMA e teste de eclosão de ovos; EHA) e in vivo (teste de redução da contagem de ovos nas fezes; TRCOF), usando o Haemonchus (H.) contortus como modelo experimental no protocolo de teste. Os resultados dos testes AMA, EHA e TRCOF foram analisados estatisticamente por meio de regressão linear e teste de Duncan. No teste AMA, na concentração de 50 mg mL-1, o percentual de mortalidade de H. contortus foi maior com o uso de A. subulatum do que com V. negundo, enquanto, no teste EHA, A. subulatum apresentou maior ação ovicida (LC50=14,2 µg mL- 1) do que V. negundo (LC50= 65,7405 µg mL-1). O TRCOF também indicou a melhor eficácia do uso de A. subulatum do que de V. negundo contra a infecção natural de parasitas gastrointestinais (GI). O extrato bruto seco das plantas utilizadas neste estudo apresentou 29,6% a 57,7% de atividade anti-helmíntica. A taxa de redução observada com o uso de A. subulatum (3 g kg-1) foi maior que com o uso de V. negundo (7 g kg-1). Em relação à análise da eficácia dos solventes utilizados para o extrato de plantas, o acetato de etila e o clorofórmio apresentaram maior ação ovicida em vermes adultos (testes in vitro), enquanto o extrato bruto metanólico aquoso apresentou maior eficácia do que os extratos brutos secos em testes in vivo. Consideramos vantajoso documentar o conhecimento indígena relativos aos procedimentos científicos padronizados, para sua validação. Este estudo irá servir de motivação para que os agricultores façam escolhas melhores referentes ao cultivo das plantas indígenas devido às suas diferentes eficácias comprovadas, servindo como alternativa para a abordagem preventiva contra as infecções parasitárias GI.
Assuntos
Doenças Parasitárias , Plantas Medicinais , Vitex/parasitologia , Amomum/parasitologia , Anti-HelmínticosRESUMO
A Mexican isolate of the nematophagous fungus Arthrobotrys musiformis was obtained from a soil sample from the Chapultepec ecological reserve zone, in Cuernavaca, Morelos, Mexico. This isolate demonstrated an important predatory activity (74.9%) against the parasitic nematode Haemonchus contortus (L3) and its fungal liquid culture filtrates (LCF) grown in two media showed the following highest nematocidal activities (NA): In Czapek-DoxBroth (CzDoxB) 80.66% and potato-dextrose broth (PDB) 49.84%. Additionally, two major compounds derived from carboxylic acids and two derivates from alkane group were identified by GC-MS. These compounds have been associated to many biological activities. On the other hand, the protein profile analysis by SDS-electrophoresis followed by a zymogram revealed a 10 kDa protein with protease activity. This study provides important information for future experiments focused to explore the potential use of this protein as well as the identified bioactive compounds presents in the LCF as potential candidates against sheep haemonchosis.
Assuntos
Ascomicetos , Haemonchus , Animais , Ovinos , Haemonchus/microbiologia , Antinematódeos , Comportamento Predatório , México , Controle Biológico de Vetores/métodos , Larva/microbiologiaRESUMO
We aimed to validate a targeted selective treatment (TST) methodology for treating parasitic gastrointestinal infections in ewes in different physiological states using parasitological and hematological parameters. Forty ewes were monitored from December 2021 to June 2022 and evaluated during various physiological stages in their life cycle. Before starting the experiment, a fecal egg count (FEC) reduction test was performed to evaluate the efficacy of the anthelmintic (AH) treatment. Weekly assessments were performed based on the Famacha© (F) system and body condition score (BCS), and ewes were subjected to AH treatment when necessary, with their physiological states recorded. Ewes were treated when they presented F ≥ 3, BCS ≤ 2.0 (when F = 2), or submandibular edema. Parasitological, i.e., FEC, and hematological, i.e., hematocrit (Ht), parameters were evaluated monthly to determine the efficiency of the TST methodology. Comparisons between the mean Ht and FEC values in ewes subjected to AH treatment and untreated ewes were performed using analysis of variance, followed by Tukey's test. Spearman's correlation was performed to determine the correlation between the variables, i.e., F scores, BCS, Ht, and FEC. All tests were performed at a significance level of 5 %. During the experimental period, 1138 evaluations were performed. The main reason for AH treatment was F ≥ 3. Ewes in early pregnancy, lactation and late pregnancy received comparatively more AH treatments than the other physiological states. Ewes in late pregnancy and lactation exhibited lower mean Ht values (23.5 % and 22.9 %) and higher mean FEC values (3269 and 1426) compared with those in early pregnancy (30.2 % and 727 EPG). In addition, a statistically significant difference was observed in the Ht and FEC values of ewes that presented submandibular edema (P < 0.001) compared with those that did not exhibit submandibular edema. The genus Haemonchus sp. showed a 96.4 % prevalence in coprocultures. A positive correlation existed between F scores and FEC (r = 0.3819) and a negative correlation between F scores and Ht (r = -0.4728). Ewes that needed AH treatment had lower mean Ht values than ewes that did not need the treatment (19.2 % × 29.3 %; P < 0.001) and higher mean FEC values (8747 × 1163; P < 0.001), confirming that these ewes needed AH treatment. The TST methodology based on F scores, BCS, and submandibular edema could effectively identify individuals in the herd needing AH treatment, identifying 13 % additional cases requiring treatment than using only the F score criterion.
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In vitro larvicidal assays carried out previously by our research group with cubebin, dihydrocubebin and hinokinin, lignans extracted from the fruits of Piper cubeba, against Haemonchus contortus larvae showed strong action larvicidal these compounds. Hinokinin was the most active (EC50 = 0.34 µg/mL) with strong action on the cuticle of the larvae as observed by scanning electron microscopy of the L3 stage. Therefore, to understand the mechanism of action of these compounds in silico studies were carried out using the enzyme phosphomethyltransferase of Haemonchus contortus that contain PMT-1 and PMT-2 di-domains responsible for phosphocholine synthesis, which is one of the main lipids in nematodes. This pathway is not found in mammals, so this enzyme is an important biological target for the development of new anthelmintics. Results of molecular docking, molecular dynamic and a density functional theory calculations studies with the three lignans show few interactions with PMT-1. However, hinokinin has important interactions with PMT-2, that can deactivate the enzyme and interrupt the phosphocholine synthesis, which is an essential compound for the development and maintenance of the nematode cuticle and its survive. Therefore, the previous results of the in vitro assay allied with in silico results, now realized; suggest that hinokinin may be a possible selective target for the development of new anthelmintics against Haemonchus contortus since the PMT-2 domain is present in this nematode.
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Anti-Helmínticos , Haemonchus , Lignanas , Nematoides , Piper , Animais , Simulação de Acoplamento Molecular , Fosforilcolina/farmacologia , Lignanas/farmacologia , Anti-Helmínticos/farmacologia , Larva , MamíferosRESUMO
Association between ovine ß-globin polymorphisms and resistance against haemonchosis was described and related to the mechanism of high oxygen affinity ßA â ßC switch during anaemia, but there are no studies regarding the involved local host responses. Phenotypic parameters and local responses were evaluated in sheep from two ß-globin haplotypes naturally infected with Haemonchus contortus. Morada Nova lambs were monitored at 63, 84 and 105 days of age for faecal egg counts and packed cell volume (PCV) under natural infection with H. contortus. At 210 days of age, lambs of Hb-AA and Hb-BB ß-globin haplotypes were euthanised, and the fundic region of abomasum was sampled for evaluation of microscopic lesions and relative expression of genes related to immune, mucin and lectin activities. Lambs harbouring the ßA allele presented an improved resistance/resilience against clinical haemonchosis, showing higher PCV during infection. Hb-AA animals presented increased eosinophilia in the abomasum compared to Hb-BB animals, accompanied by higher Th2 profile, mucin and lectin activity transcripts, while the inflammatory response was increased in Hb-BB animals. This is the first report to demonstrate an enhanced local response in the primary site of H. contortus infection related to ßA allele of ß-globin haplotype.
Assuntos
Hemoncose , Haemonchus , Doenças dos Ovinos , Animais , Ovinos , Haemonchus/genética , Hematócrito/veterinária , Mucinas/genética , Lectinas , Fezes , Contagem de Ovos de Parasitas/veterináriaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Gastrointestinal nematodes (GIN) control in small ruminants has relied on the systematic use of synthetic anthelmintics (AH), their effectiveness has been progressively decreasing due to the rise and diffusion of anthelmintic resistances. The most prevalent genera affecting small ruminants were Haemonchus spp., and Trichostrongylus spp. The investigation of new anthelmintics in plants is a highly studied option, especially when it is linked to ethnobotanical knowledge and phenolic compounds. THE AIMS OF OUR STUDY: Four medicinal plants mentioned in traditional medicine were selected to evaluate their anthelmintic proprieties at different stages of the life cycle of GIN, namely: Kyllinga odorata Valh., Cassia occidentalis L., Artemisia absinthium L, and Verbena litoralis Kunth and to explore the role of polyphenols in the AH activity. MATERIALS AND METHODS: To evaluate the anthelmintic activity in this study, two models of GIN species, namely Haemonchus contortus (Hc) and Trichostrongylus colubriformis (Tc) were selected and tested on two in vitro assays: 1) Larval Exsheathment Inhibition Assay (LEIA) and, 2) Egg Hatch Assay (EHA). To explore the role of tannins and polyphenols in AH activity by comparing the effects of LEIA and EHA with or without polyvinylpolypyrrolidone (PVPP) and to characterize the phytochemical composition of the most active plants using ultra-high performance liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS). RESULTS: C. occidentalis exhibited the highest activity on LEIA (EC50 = 250.42-41.80 µg/mL) and A. absinthium on egg hatching processes (EC50 = 121.70-137.34 µg/mL) in both species of GIN. The inhibition in the development of eggs was from 67.70% to 96.36% on H. contortus, and from 78.87% to 99.65% on T. colubriformis. At the maximal dose, Additionally, it was observed that the AH on eggs varies according to the GIN species: on H. contortus the extracts tested blocked the formation of larvae Ovicidal Effect (% higher OE) and on T. colubriformis they blocked the appearance of L1 larvae, Larvae Failing Eclosion (% higher LFE). After PVPP, a reduction in AH activity on LEIA and EHA was noted, especially with C. occidentalis (87.20-67.00% of larvae exsheathment, (p < 0.05) and 40.51-24.96% of egg hatching, (p > 0.05) of both parasite species. Nine putative features were identified using HRMS and MS/MS after addition of PVPP. CONCLUSIONS: The present study demonstrated that C. occidentalis, A. absinthium, and K. odorata, which parts have been traditionally used as medicinal plants are a valuable source of active compounds with anthelmintic activity. The medicinal use of these plants against GIN parasites was proven by in vitro analysis. Therefore exploration of the secondary metabolites of these plant extracts and testing of isolated fractions of active compounds under in vivo experiments are planned and represent a specific challenge for alternative drug research. Regarding the PVPP, in this study we hypotheses about the standard doses it was not able to completely absorb the polyphenols of extracts of K. odorata, C. occidentalis, and A. absinthium, which would lead to more studies to evaluate the role of this product in the absorption of phenolic compounds.
Assuntos
Anti-Helmínticos , Haemonchus , Plantas Medicinais , Animais , Polifenóis/farmacologia , Espectrometria de Massas em Tandem , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Anti-Helmínticos/farmacologia , Anti-Helmínticos/química , Fenóis/farmacologia , Ruminantes/parasitologia , LarvaRESUMO
Citrus fruits are consumed all over the world and their by-products are used for animal feed and essential oils production. This study aimed to evaluate the in vitro and in vivo activity of Citrus aurantium var. Dulcis essential oil (CaEO) combined with ABZ against benzimidazole resistant Haemonchus contortus. In vitro egg hatching assays (EHA) were performed using CaEO and ABZ to estimate the effective concentration to achieve 50% egg death (EC50) values and calculate the test essential oil and drug combinations using a simplex-centroid mixture design. These concentrations were used for a second round of EHAs. Sixteen sheep were randomly allocated into two groups and treated with ABZ and the combination of CaEO and ABZ, and faecal egg count reduction tests were performed. In the first round of EHA, CaEO and ABZ showed EC50 values of 0.57 and 0.0048 mg mL-1, respectively. The H. contortus strain used in the study was shown to be highly benzimidazole resistant, with only 1.5% of parasites having susceptible ß-tubulin SNP genotypes. The ABZ reduced the shedding of nematode eggs by 78%, however, its combination with CaEO reduced faecal egg counts by only 9%. The present study is important to highlight the interferences of natural products in anthelmintic metabolism and consequently in drug efficacy.
Assuntos
Anti-Helmínticos , Citrus , Hemoncose , Haemonchus , Nematoides , Óleos Voláteis , Doenças dos Ovinos , Animais , Ovinos , Albendazol/farmacologia , Albendazol/uso terapêutico , Óleos Voláteis/farmacologia , Óleos Voláteis/uso terapêutico , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Benzimidazóis/farmacologia , Fezes/parasitologia , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/parasitologia , Contagem de Ovos de Parasitas/veterinária , Resistência a Medicamentos , Hemoncose/tratamento farmacológico , Hemoncose/veterinária , Hemoncose/parasitologiaRESUMO
Haemonchus contortus (Hc) is an important parasitic nematode of small ruminants. In this study we assembled the transcriptome of Hc as a model to contribute to the knowledge about the profile of the differential gene expression between two Mexican Hc strains under different anthelmintic resistance statuses, one susceptible and the other resistant to ivermectin (IVMs and IVMr, respectively), in order to improve and/or to have new strategies of control and diagnosis. The transcript sequence reads were assembled and annotated. Overall, ~127 Mbp were assembled and distributed into 77,422 transcript sequences, and 4394 transcripts of the de novo transcriptome were matched base on at least one of the following criteria: (1) Phylum Nemathelminthes and Platyhelminthes, important for animal health care, and (2) ≥55% of sequence identity with other organisms. The gene ontology (GO) enrichment analysis (GOEA) was performed to study the level of gene regulation to IVMr and IVMs strains using Log Fold Change (LFC) filtering values ≥ 1 and ≥ 2. The upregulated-displayed genes obtained via GOEA were: 1993 (for LFC ≥ 1) and 1241 (for LFC ≥ 2) in IVMr and 1929 (for LFC ≥ 1) and 835 (for LFC ≥ 2) in IVMs. The enriched GO terms upregulated per category identified the intracellular structure, intracellular membrane-bounded organelle and integral component of the cell membrane as some principal cellular components. Meanwhile, efflux transmembrane transporter activity, ABC-type xenobiotic transporter activity and ATPase-coupled transmembrane transporter activity were associated with molecular function. Responses to nematicide activity, pharyngeal pumping and positive regulation of synaptic assembly were classified as biological processes that might be involved in events related to the anthelmintic resistance (AR) and nematode biology. The filtering analysis of both LFC values showed similar genes related to AR. This study deepens our knowledge about the mechanisms behind the processes of H. contortus in order to help in tool production and to facilitate the reduction of AR and promote the development of other control strategies, such as anthelmintic drug targets and vaccines.
RESUMO
This study aimed to evaluate the effects of different doses of quebracho extract tannins (Schinopsis lorenzii) (EQ) included in the basal diet (DB) on the count of eggs per gram of feces (EPG), count of parasites in the abomasum, hematological, and immunological parameters in lambs naturally infected with Haemonchus contortus. Forty-eight lambs were evaluated for 42 days and divided into four groups (n = 12): CONT- lambs that received DB without EQ supplementation; G1%- DB with EQ at 1% of dry matter intake (DMI) level; G3%- DB with EQ at the 3% of DMI level; G6%- DB with EQ at the 6% DMI level. For EPG counts, feces were collected weekly. For counting abomasal parasites, at the end of the experiment (D42) half of the lambs in each group were slaughtered. For hematological and immunological analysis, venous blood samples were collected at D0, D21, and D42 to perform the erythrocyte count, mean cell volume (MCV), hematocrit, differential leukocyte count, total plasma protein dosage, and serum IgA class anti-H. contortus using an indirect enzyme immunoassay (ELISA). In the EPG count, there was no significant difference (p > 0.05) between the groups compared with the control. Analysis of the abomasal parasite count showed Haemonchus contortus in its entirety; however, there was no significant difference (p > 0.05) in the number of males, females, and total parasites. No differences (p > 0.05) were observed in erythrocyte count, MCV, hematocrit, and total plasma protein. The average total leukocyte, monocyte, eosinophil, and basophil counts showed no difference (p > 0.05). However, the averages of segmented neutrophils in the G1% and G3% groups presented a reduction in the percentage of cells (p < 0.05) between D21 and D42. Regarding the lymphocyte count, a difference was observed in the CONT and G3% groups at D21 and D42 (p < 0.05), when compared to D0. The analyses of the average absorbance of anti-Haemonchus contortus serum IgA showed no difference (p > 0.05), when the groups were analyzed over time or when compared between them. It can be concluded that the inclusion of different doses of tannins from EQ did not alter the parasitological, hematological, and immunological parameters of lambs infected with H. contortus.
Assuntos
Hemoncose , Haemonchus , Doenças Parasitárias , Doenças dos Ovinos , Feminino , Masculino , Ovinos , Animais , Taninos , Carneiro Doméstico , Proteínas Sanguíneas , Imunoglobulina A , Hemoncose/tratamento farmacológico , Hemoncose/veterináriaRESUMO
Artemisia cina is a plant used in traditional Chinese medicine as a remedy for parasitic diseases. This study describes the isolation and chemical characterization of anthelmintic compounds of A. cina against Haemonchus contortus infective larvae (L3) through lethal testing. Previously, three extracts-n-hexane (HexAc), ethyl acetate (EtOAc) and methanol (MeOAc)-were evaluated at concentrations of 4 to 0.5 mg/mL, resulting in the HexAc extract with the greatest effect of 76.6% mortality of the larvae at 4 mg/mL. Then, this was chemically fractioned by polarity, obtaining seven fractions (C1F1-C1F7), and, when evaluated at concentrations from 2 to 0.25 mg/mL, the 2 mg/mL C1F5 fraction produced an effect against the nematode H. contortus of 100% mortality of the larvae. Thus, this fraction was fractionated again by column chromatography, obtaining twelve subfractions (C2F1-C2F12) which were evaluated from 1 to 0.125 mg/mL, with the C2F5 subfraction causing a nematicidal effect of 100% mortality. NMR analysis of one (1H, 13C and DEPT) and two dimensions (COSY, HSQC and HMBC) and mass spectrometry of this fraction allowed us to identify the mixture of 3'-demethoxy-6-O-demethylisoguaiacin and norisoguaiacin. Therefore, it can be assumed that the mixture of these compounds is responsible for the anthelmintic effect. These results indicate that A. cina containing anthelmintic compounds and might be used as an antiparasitic drug against H. contortus.