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1.
Exp Parasitol ; 225: 108105, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33812980

RESUMO

Following the previous findings reported by the present authors on the anthelmintic effect of hydro-ethanolic extract of Mentha pulegium, the volatile constituents of M. pulegium are now assessed in the present study by exploring its anthelmintic and its antioxidant proprieties using in vitro and in vivo assays. Egg hatch assay (EHA) and adult worm's motility assays (AWMA) were used to assess the in vitro activity against Haemonchus. contortus. The in vivo anthelmintic potential was evaluated in mice infected with Heligmosomoides polygyrus using faecal egg count reduction (FECR) and total worm count reduction (TWCR). M. pulegium EO demonstrated 100% inhibition in the EHA at 200 µg/mL (IC50 = 56.36 µg/mL). In the AWM assay, EO achieved total worms paralysis 6 h after treatment exposure. This nematicidal effect was associated to morphological damages observed in the cuticular's worm using environmental scanning electron microscopy (ESEM). At 400 mg/kg, M. pulegium oil showed 75.66% of FECR and 80.23% of TWCR. The antioxidant potential of this plant was also monitored by several in vitro assays: total antioxidant capacity was 205.22 mg GAE/g DW, DPPH quenching effect was IC50 = 140 µg/mL, ABTS activity IC50 = 155 µg/mL and FRAP effect of 660 µg/mL. Regarding the in vivo assay, M. pulegium EO demonstrated a protective effect against oxidative stress by increasing the activity of the endogenous antioxidants (SOD, CAT and GPx) during H. polygyrus infection.


Assuntos
Anti-Helmínticos/farmacologia , Mentha pulegium/química , Óleos Voláteis/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Anti-Helmínticos/análise , Anti-Helmínticos/uso terapêutico , Antioxidantes/metabolismo , Modelos Animais de Doenças , Hemoncose/tratamento farmacológico , Hemoncose/parasitologia , Haemonchus/efeitos dos fármacos , Camundongos , Óleos Voláteis/análise , Óleos Voláteis/uso terapêutico , Óvulo/efeitos dos fármacos , Carga Parasitária
2.
Acta Trop ; 219: 105920, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33861973

RESUMO

The synthesis of thiophenic compounds, previously identified in Tagetes patula, revealed that 4-(5'-(hydroxymethyl)-[2,2'-bithiophene]-5-yl)but-3-yn-1-ol), or simply Thio1, has a pronounced in vitro anthelmintic effect against Haemonchus contortus, showing 100% efficacy in the egg hatch and larval development tests presenting EC50 = 0.1731 mg.mL-1 and EC50 = 0.3243 mg.mL-1, respectively. So, this compound was selected to preparation of a nanostructured formulation to be orally administered to Santa Inês sheep. In general, from the fecal egg count reduction test (FECRT), it was observed that the product kept the parasitic load in the digestive tract of the hosts stable, with eggs per gram of faeces (EPG) counts having a mean value < 3,000 (EPGmean = 2167.1, efficacy = 36,45%), thus protecting the animals from health risks caused by a massive nematode infestation. To better understand the mode of action of this thiophene derivative, in silico molecular modeling studies were carried out with the glutamate-activated chloride channel (GluCl), a well-known molecular target of anthelmintic compounds. Based on the affinity score (GlideScore = -5.7 kcal.mol-1) and the proposed binding mode, Thio1 could be classified as a potential GluCl ligand, justifying the promising results observed in the anthelmintic assays.


Assuntos
Haemonchus/efeitos dos fármacos , Nanoestruturas/química , Tiofenos/química , Tiofenos/farmacologia , Animais , Anti-Helmínticos/química , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Asteraceae/química , Composição de Medicamentos , Fezes/parasitologia , Hemoncose/tratamento farmacológico , Haemonchus/fisiologia , Contagem de Ovos de Parasitas/veterinária , Ovinos , Doenças dos Ovinos/parasitologia , Tiofenos/uso terapêutico
3.
Acta Trop ; 217: 105869, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33631121

RESUMO

Haemonchus contortus, a blood-sucking parasite of small ruminants, produces very important economic losses in the productive sector. This abomasum parasite has become resistant to most commercial drugs worldwide, and alternatives to fight this problem are urgently needed. Essential oils (EO) are a complex mixture of volatile secondary metabolites, composed mainly by terpenoids and phenolic compounds, from plants that have several pharmacological properties, including anthelmintic activity. Particularly, citrus peel is a source of cold-pressed EO, where limonene is its major component, and can be used as an additional food component for ruminants. The aim of the present work was to determine the in vitro anthelmintic activity of EO from Citrus bergamia (EOB), C. x paradisii (EOG) and limonene against the benzimidazole-susceptible Kirby isolate of H. contortus, using the egg hatch test (EHT) and the exsheathed third stage larval motility test (XLMT) using a WMicroTracker equipment. Albendazole (ABZ) and monepantel (MON) were used as positive controls. The 50% inhibitory concentrations (IC50) in XLMT were 8.77 and 13.88 µg/ml for EOB and EOG respectively, after an incubation of 72 h. An interesting observation on XLMT resulted when the positive controls were tested on the same plate, but in different well of the EOB. The volatile components of the EO significantly influenced (P < 0.05) the percentage of larval motility, reducing values from 66.9 to 19.6% for ABZ, and from 72.8 to 33.7% for MON, when comparing the activity of positive controls in a control plate without EO. The in vitro anthelmintic activity of EOB and EOG shows that they could be interesting candidates for nematode control. It is still necessary additional studies against the adult stage of H. contortus in efficacy trials in infected animals to validate their anthelmintic activity.


Assuntos
Anti-Helmínticos/farmacologia , Citrus/química , Haemonchus/efeitos dos fármacos , Óleos Voláteis/farmacologia , Animais , Larva/efeitos dos fármacos
4.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33431676

RESUMO

Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.


Assuntos
Búfalos/imunologia , Resistência à Doença , Hemoncose/microbiologia , Pulmão/imunologia , Linfonodos/imunologia , Tricostrongilose/microbiologia , Tuberculose Bovina/microbiologia , Animais , Antinematódeos/farmacologia , Búfalos/microbiologia , Búfalos/parasitologia , Bovinos , Coinfecção , Progressão da Doença , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Eosinófilos/microbiologia , Eosinófilos/parasitologia , Fezes/parasitologia , Feminino , Fenbendazol/farmacologia , Hemoncose/tratamento farmacológico , Hemoncose/mortalidade , Hemoncose/parasitologia , Haemonchus/efeitos dos fármacos , Haemonchus/genética , Haemonchus/patogenicidade , Imunoglobulina A/sangue , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/parasitologia , Linfonodos/efeitos dos fármacos , Linfonodos/microbiologia , Linfonodos/parasitologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/microbiologia , Mastócitos/parasitologia , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/patogenicidade , Índice de Gravidade de Doença , Análise de Sobrevida , Tricostrongilose/tratamento farmacológico , Tricostrongilose/mortalidade , Tricostrongilose/parasitologia , Trichostrongylus/efeitos dos fármacos , Trichostrongylus/genética , Trichostrongylus/patogenicidade , Tuberculose Bovina/tratamento farmacológico , Tuberculose Bovina/mortalidade , Tuberculose Bovina/parasitologia
5.
J Parasitol ; 107(1): 23-28, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33498082

RESUMO

Gastrointestinal nematode infection of small ruminants causes losses in livestock production. Plant compounds show promises as alternatives to commercial anthelmintics that have been exerting selective pressures that lead to the development of drug-resistant parasites. Soybean (Glycine max) is an economical value crop, with a higher protein content compared to other legumes. The objective of this study was to evaluate whether the protease inhibitors exuded from the G. max mature seeds have anthelmintic activity against Haemonchus contortus. To obtain the soybean exudates (SEX), mature seeds were immersed in 100 mM sodium acetate buffer, pH 5.0, at 10 C, for 24 hr. Then the naturally released substances present in SEX were collected and exhaustively dialyzed (cutoff 12 kDa) against distilled water. The dialyzed seed exudates (SEXD) were heated at 100 C for 10 min and centrifuged (12,000 g, at 4 C for 15 min). The supernatant obtained was recovered and designated as the heat-treated exudate fraction (SEXDH). The protein content, protease inhibitor activity, and the effect of each fraction on H. contortus egg hatch rate were evaluated. The inhibition extent of SEX, SEXD, and SEXDH on H. contortus egg proteases was 31.1, 42.9, and 63.8%, respectively. Moreover, SEX, SEXD, and SEXDH inhibited the egg hatching with EC50 of 0.175, 0.175, and 0.241 mg ml-1, respectively. Among the commercial protease inhibitors tested, only EDTA and E-64 inhibited the H. contortus hatch rate (79.0 and 28.9%, respectively). We present evidence demonstrating that soybean exudate proteins can effectively inhibit H. contortus egg hatching. This bioactivity is displayed by thermostable proteins and provides evidence that protease inhibitors are a potential candidate for anthelmintic use.


Assuntos
Exsudatos e Transudatos/química , Haemonchus/efeitos dos fármacos , Extratos Vegetais/farmacologia , Inibidores de Proteases/farmacologia , Sementes/química , Soja/química , Animais , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/enzimologia , Haemonchus/fisiologia , Concentração de Íons de Hidrogênio , Peptídeo Hidrolases/isolamento & purificação , Extratos Vegetais/isolamento & purificação , Inibidores de Proteases/isolamento & purificação , Ovinos , Doenças dos Ovinos/parasitologia , Proteínas de Soja/química
6.
J Ethnopharmacol ; 265: 113142, 2021 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-32697959

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: The leaf of Sarcocephalus latifolius is known to be used traditionally by the Fulanis in Nigeria to deworm animals. As helminthosis remains a major constraint to profitable livestock production worldwide, a precarious situation aggravated by the advent of resistant parasites, the discovery of new anthelmintics is a priority, necessitating exploration of medicinal plants for their anthelmintic principles. AIM OF THE STUDY: To identify and characterise compounds with anthelmintic activity from the leaf of Sarcocephalus latifolius. MATERIALS AND METHODS: Powdered S. latifolius leaves were extracted by successive maceration with n-hexane, chloroform and acetone. The dried extracts were evaluated for anthelmintic activity against Haemonchus placei adult worms, and the most active extract was subjected to bioassay-guided chromatographic separations. The isolated compounds were evaluated for cytotoxicity against the mammalian HeLa and MC3T3-E1 cell lines, using alamar blue and CellTitreGloTM to quantify cell viability. LC50 values were computed from the in vitro anthelmintic activity data by fitting to a non-linear regression equation (variable slope). Isolated compounds were characterized using spectroscopic and mass spectrometric analyses. RESULTS: Anthelmintic activity LC50 values for n-hexane, chloroform and acetone extracts were 47.85, 35.76 and 5.72 (mg/mL), respectively. Chromatographic separation of acetone extract afforded two bioactive epimers, identified as vincosamide (LC50 14.7 mg/mL) and strictosamide (LC50 12.8 mg/mL). Cytotoxicity evaluation showed that, below 200 µg/mL (400 µM), neither compound was toxic to the HeLa or MC3T3-E1 cells. CONCLUSION: Vincosamide and strictosamide could serve as novel scaffolds for the development of anthelmintic derivatives with improved potency and helminth selectivity.


Assuntos
Anti-Helmínticos/farmacologia , Alcaloides Indólicos/farmacologia , Rubiaceae/química , Alcaloides de Vinca/farmacologia , Células 3T3 , Animais , Anti-Helmínticos/isolamento & purificação , Anti-Helmínticos/toxicidade , Haemonchus/efeitos dos fármacos , Células HeLa , Humanos , Alcaloides Indólicos/isolamento & purificação , Alcaloides Indólicos/toxicidade , Dose Letal Mediana , Camundongos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Extratos Vegetais/toxicidade , Folhas de Planta , Alcaloides de Vinca/isolamento & purificação , Alcaloides de Vinca/toxicidade
7.
J Med Chem ; 64(1): 840-844, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33352050

RESUMO

A series of 1-methyl-1H-pyrazole-5-carboxamides were synthesized as potent inhibitors of the parasitic nematode of sheep, Haemonchus contortus. These compounds did not show overt cytotoxicity to a range of mammalian cell lines under standard in vitro culture conditions, had high selectivity indices, and were progressed to an acute toxicity study in a rodent model. Strikingly, acute toxicity was observed in mice. Experiments measuring cellular respiration showed a dose-dependent inhibition of mitochondrial respiration. Under these conditions, potent cytotoxicity was observed for these compounds in rat hepatocytes suggesting that the potent acute mammalian toxicity of this chemotype is most likely associated with respiratory inhibition. In contrast, parasite toxicity was not correlated to acute toxicity or cytotoxicity in respiring cells. This paper highlights the importance of identifying an appropriate in vitro predictor of in vivo toxicity early on in the drug discovery pipeline, in particular assessment for in vitro mitochondrial toxicity.


Assuntos
Antiprotozoários/farmacologia , Haemonchus/efeitos dos fármacos , Pirazóis/química , Animais , Antiprotozoários/química , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Feminino , Masculino , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Pirazóis/farmacologia , Ratos , Ovinos/parasitologia , Relação Estrutura-Atividade
8.
Trends Parasitol ; 37(3): 240-250, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33317926

RESUMO

Anthelmintic drugs are the major line of defense against parasitic nematode infections, but the arsenal is limited and resistance threatens sustained efficacy of the available drugs. Discoveries of the modes of action of these drugs and mechanisms of resistance have predominantly come from studies of a related nonparasitic nematode species, Caenorhabditis elegans, and the parasitic nematode Haemonchus contortus. Here, we discuss how our understanding of anthelmintic resistance and modes of action came from the interplay of results from each of these species. We argue that this 'cycle of discovery', where results from one species inform the design of experiments in the other, can use the complementary strengths of both to understand anthelmintic modes of action and mechanisms of resistance.


Assuntos
Resistência a Medicamentos , Nematoides/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Anti-Helmínticos/uso terapêutico , Caenorhabditis elegans/efeitos dos fármacos , Haemonchus/efeitos dos fármacos , Infecções por Nematoides/terapia , Pesquisa/normas , Pesquisa/tendências
9.
Exp Parasitol ; 218: 108004, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32961172

RESUMO

Haemonchus contortus is a hematophagous parasite causing damage to the production of ruminant animals throughout the world. This study evaluated the in vitro effect of proteins from Moringa oleifera (WSMoL - Water Soluble M. oleifera Lectin and cMoL - coagulant M. oleifera Lectin) on the motility of infective larvae and adult male and female worms of H. contortus. The specific activity of total proteases and the morphology of the worms exposed to the lectins were observed. Both lectins inhibited motility of all parasite stages tested. WSMoL and cMoL at 500 µg mL-1 interfered in the motility of larvae. Values of 11.1% and 8.1% were the lowest motility indices of larvae with sheath, and 30.6% and 16.4% were the lowest motility indices of exsheathed larvae treated with WSMoL and cMoL, respectively. In 1 mg mL-1 solutions of WSMoL and of cMoL, the motility index of adult male worms was 23.3% (p < 0.001) and 20% (p < 0.001), while the motility index of adult female worms was 63.3% (p > 0.05) and 26.6% (p < 0.001), respectively. Greater proteolytic activity was detected in extracts obtained from adult worms, male and female, after incubation with the lectins. Morphological changes caused by the lectins were revealed by changes in the crests of the cuticle, in the longitudinal striations and at the vulva.


Assuntos
Haemonchus/efeitos dos fármacos , Moringa oleifera/química , Lectinas de Plantas/farmacologia , Sementes/química , Animais , Feminino , Haemonchus/enzimologia , Haemonchus/fisiologia , Haemonchus/ultraestrutura , Larva/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Varredura , Movimento/efeitos dos fármacos , Peptídeo Hidrolases/metabolismo , Lectinas de Plantas/administração & dosagem , Lectinas de Plantas/isolamento & purificação
10.
Exp Parasitol ; 218: 107980, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32877640

RESUMO

Acacia farnesiana pods are rich in secondary metabolites and their biological activities have been recorded as antibacterial, antioxidant and anthelmintic. Previously, an in vitro bioguided study showed the important ovicidal and larvicidal effects of an organic fraction (EtOAc-F) from a hydroalcoholic extract of A. farnesiana pods against Haemonchus contortus. The present study aimed to assess the in vivo anthelmintic effect of EtOAc-F from A. farnesiana pods on the H. contortus faecal egg elimination in female lambs and on the infective larvae (L3) population reduction in coprocultures. The EtOAc-F was obtained from a hydroalcoholic extract from A. farnesiana pods through chromatographic procedures; additionally, some secondary compounds were identified using high-performance liquid chromatography (HPLC). Twenty-one 'Katahdin' crossbred female lambs ranging from three to four months of age, with body weights 21.9 ± 0.39 kg were used. Animals were orally infected with H. contortus (L3) by a single dose of 350 L3/kg BW. Three experimental groups (n = 7) were assigned as follows: 1) Control (untreated), 2) Albendazole, as a positive control (at 7.5 mg/kg BW, unique dose) and 3) EtOAc-F (at 100 mg/kg BW, once every third day, with three applications in total). Individual faecal samples were collected once a week for 5 weeks (at days 38, 45, 52, 59 and 66) post-treatment, to measure the faecal egg counts (FEC) and to obtain the H. contortus (L3) population from faecal cultures. The highest FEC reduction caused by EtOAc-F was 67.7%; meanwhile, albendazole showed a total FEC reduction after the second week post-treatment (day 45). On the other hand, the fraction caused an important reduction in the larval population in coprocultures (54.3-68.5%). The phytochemical analysis revealed the presence of galloyl derivatives and flavonoids as major compounds. The A. farnesiana pods could serve as a natural anthelmintic for the control of H. contortus, and perhaps for controlling other parasites of veterinary importance.


Assuntos
Acacia/química , Anti-Helmínticos/uso terapêutico , Hemoncose/veterinária , Extratos Vegetais/uso terapêutico , Doenças dos Ovinos/tratamento farmacológico , Animais , Anti-Helmínticos/isolamento & purificação , Anti-Helmínticos/farmacologia , Fezes/parasitologia , Feminino , Flavonoides/química , Ácido Gálico/química , Hemoncose/tratamento farmacológico , Hemoncose/parasitologia , Haemonchus/efeitos dos fármacos , Haemonchus/isolamento & purificação , Hematócrito/veterinária , Contagem de Ovos de Parasitas/veterinária , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Distribuição Aleatória , Ovinos , Doenças dos Ovinos/parasitologia
11.
Parasitol Res ; 119(9): 2851-2862, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32651637

RESUMO

The field strain of Haemonchus contortus has a long history of anthelmintic resistance. To understand this phenomenon, the benzimidazole resistance profile was characterized from the Malaysian field-resistant strain by integrating phenotypic, genotypic and proteomic approaches. The faecal egg count reduction test (FECRT) demonstrated that benzimidazole resistance was at a critical level in the studied strain. The primary resistance mechanism was attributed to F200Y mutation in the isotype 1 ß-tubulin gene as revealed by AS-PCR and direct sequencing. Furthermore, the protein response of the resistant strain towards benzimidazole (i.e., albendazole) treatment was investigated via two-dimensional difference gel electrophoresis (2D-DIGE) and tandem liquid chromatography-mass spectrometry (LC-MS/MS). These investigations illustrated an up-regulation of antioxidant (i.e., ATP-binding region and heat-shock protein 90, superoxide dismutase) and metabolic (i.e., glutamate dehydrogenase) enzymes and down-regulation of glutathione S-transferase, malate dehydrogenase, and other structural and cytoskeletal proteins (i.e., actin, troponin T). Findings from this study are pivotal in updating the current knowledge on anthelmintic resistance and providing new insights into the defence mechanisms of resistant nematodes towards drug treatment.


Assuntos
Albendazol/farmacologia , Anti-Helmínticos/farmacologia , Benzimidazóis/farmacologia , Resistência a Medicamentos/genética , Haemonchus/efeitos dos fármacos , Animais , Antioxidantes/metabolismo , Cromatografia Líquida , Glutamato Desidrogenase/metabolismo , Hemoncose/tratamento farmacológico , Haemonchus/genética , Reação em Cadeia da Polimerase , Proteômica , Ovinos , Doenças dos Ovinos/parasitologia , Espectrometria de Massas em Tandem , Tubulina (Proteína)/genética
12.
Exp Parasitol ; 217: 107957, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32687847

RESUMO

The ruminant livestock production sector is under threat due to the infections with gastrointestinal nematode parasites and the subsequent development of anthelmintic resistance. One of most common and pathogenic species in small ruminants is Haemonchus contortus. The ability to control the infections with this and other gastrointestinal nematodes relies heavily on the use of anthelmintic drugs. Although resistance to all major classes of anthelmintics has been shown in H. contortus, the precise mechanism of resistance acquisition is only known for benzimidazoles. F200Y (TAC) is a common point mutation in the isotype 1 ß tubulin gene which is associated with an effective increase in the resistance towards benzimidazole drugs. Here, we show the utility of using this mutation as a marker in a droplet digital PCR assay to track how two H. contortus laboratory strains, characterized by different resistance levels, change with respect to this mutation, when subjected to increasing concentrations of thiabendazole. Additionally, we wanted to investigate whether exposure to a discriminating dose of thiabendazole in the egg hatch test resulted in the death of all H. contortus eggs with a susceptible genotype. We found the MHco5 strain to maintain an overall higher frequency of the F200Y mutation (80-100%) over all drug concentrations, whilst a steady, gradual increase from around 30%-60% was observed in the case of the MHco4 strain. This is further supported by the dose-response curves, displaying a much higher tolerance of the MHco5 strain (LD50 = 0.38 µg/ml) in comparison to the MHco4 strain (LD50 = 0.07 µg/ml) to the effects of thiabendazole. All things considered, we show that the F200Y mutation is still a viable and reliable marker for the detection and surveillance of benzimidazole drug resistance in H. contortus in Europe.


Assuntos
Anti-Helmínticos/farmacologia , Haemonchus/genética , Taxa de Mutação , Tiabendazol/farmacologia , Tubulina (Proteína)/genética , Animais , DNA de Helmintos/isolamento & purificação , Relação Dose-Resposta a Droga , Resistência a Medicamentos/genética , Frequência do Gene , Marcadores Genéticos , Genótipo , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/classificação , Haemonchus/efeitos dos fármacos , Dose Letal Mediana , Óvulo/efeitos dos fármacos , Fenótipo , Mutação Puntual , Reação em Cadeia da Polimerase , Ovinos , Doenças dos Ovinos/parasitologia
13.
PLoS One ; 15(6): e0235072, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32574178

RESUMO

The abomasal parasitic nematode Haemonchus contortus can influence the abomasal microbiome of the host. On the other hand, no information occurs on the influence of the parasite on the hindgut microbiome of the host. We evaluated the impact of Haemonchus contortus on the fecal microbial community of the experimentally infected lambs treated with a mixture of medicinal herbs to ameliorate the haemonchosis. Twenty-four female lambs were divided into four groups: infected animals (Inf), infected animals supplemented with a blend of medicinal herbs (Inf+Herb), uninfected control animals (Control), and uninfected animals supplemented with medicinal herbs (C+Herb). Inf and Inf+Herb lambs were infected orally with approximately 5000 L3 larvae of a strain of H. contortus susceptible to anthelmintics (MHco1). Herb blend (Herbmix) consisted of dry medicinal plants of Althaea officinalis, Petasites hybridus, Inula helenium, Malva sylvestris, Chamomilla recutita, Plantago lanceolata, Rosmarinus officinalis, Solidago virgaurea, Fumaria officinalis, Hyssopus officinalis, Melisa officinalis, Foeniculum vulgare, and Artemisia absinthium. Each animal was fed meadow hay and a commercial concentrate (600 + 350 g DM/d). Inf+Herb and C+Herb lambs were fed Herbmix (100 g DM/d and animal). Treatment lasted for 50 days. The fecal microbial fermentation parameters (short-chain fatty acids, ammonia, and pH) were evaluated at intervals of 0, 20, 32, and 50 days. The fecal eubacterial populations were evaluated by denaturing gradient gel electrophoresis (DGGE) at day 32 when H. contortus infection was the highest. No substantial effects of the H. contortus infection and the herbal treatment on fecal microbial fermentation parameters and fecal eubacterial populations were observed. Evaluation of DGGE patterns by Principal component analysis pointed to the tendency to branch the C+Herb group from the other experimental groups on Day 32. The results indicate that hindgut microbial activity was not disturbed by H. contortus infection and herbal treatment.


Assuntos
Abomaso/efeitos dos fármacos , Sistema Digestório/efeitos dos fármacos , Hemoncose/tratamento farmacológico , Haemonchus/efeitos dos fármacos , Preparações de Plantas/farmacologia , Plantas Medicinais/química , Doenças dos Ovinos/tratamento farmacológico , Abomaso/microbiologia , Abomaso/parasitologia , Animais , Anti-Helmínticos/farmacologia , Sistema Digestório/microbiologia , Sistema Digestório/parasitologia , Quimioterapia Combinada , Fezes/microbiologia , Fezes/parasitologia , Feminino , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/fisiologia , Microbiota/efeitos dos fármacos , Microbiota/fisiologia , Fitoterapia/métodos , Plantas Medicinais/classificação , Ovinos , Doenças dos Ovinos/parasitologia , Especificidade da Espécie
14.
J Helminthol ; 94: e135, 2020 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-32127057

RESUMO

Haemonchus contortus is a haematophagous gastrointestinal nematode (GIN) that causes severe anaemia and even death in small ruminants, negatively impacting the economic viability of farms. Traditionally, this parasite has been controlled with chemical compounds; however, inadequate use of these types of products has favoured the emergence of anthelmintic resistance. Therefore, it is necessary to search for alternatives for GIN control. Previous studies have reported the anthelmintic activity of edible mushroom extracts against H. contortus. A recent study reported that a fraction constituted of different fatty acids and ß-sitosterol isolated from the basidiomata of the edible mushroom Pleurotus djamor ECS-123 has ovicidal and larvicidal activity against H. contortus. Thus, this study aimed to assess the anthelmintic activity of the pure molecules: pentadecanoic acid, palmitic acid, ß-sitosterol, stearic acid and linoleic acid. For this purpose, an egg-hatching inhibition test was carried out in which the compounds were evaluated individually and in combination at a final concentration of 20 mg mL-1. Furthermore, larval mortality was assessed using a combination of the five commercial compounds previously mentioned at different concentrations (1.25-20 mg mL-1). Palmitic acid and stearic acid, in some combinations, inhibited H. contortus egg hatching by 100%. On the other hand, in the larval mortality test, the combination of the five compounds showed dose-dependent behaviour, and 100% mortality was obtained 24 h post-incubation. Pure molecules and their combinations have anthelmintic-like activity against the eggs and larvae of H. contortus.


Assuntos
Antinematódeos/farmacologia , Ácidos Graxos/farmacologia , Haemonchus/efeitos dos fármacos , Sitosteroides/farmacologia , Agaricales/química , Animais , Bioensaio , Ácidos Graxos/química , Ácidos Graxos/classificação , Trato Gastrointestinal/parasitologia , Larva/efeitos dos fármacos , Masculino , Extratos Vegetais/farmacologia , Ovinos/parasitologia
15.
Parasit Vectors ; 13(1): 114, 2020 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-32122383

RESUMO

BACKGROUND: Benzimidazole (BZ) resistance in gastrointestinal nematodes is a worldwide problem for livestock production, particularly in small ruminants. Assignment of the emergence of resistance using sensitive and reliable methods is required to adopt the correct strategies for control. In Sudan, BZ resistant Haemonchus contortus populations were recently reported in goats in South Darfur. This study aimed to provide additional data regarding albendazole efficacy and to describe the prevailing molecular BZ resistance mechanisms. METHODS: Faecal egg count reduction and egg hatch tests (EHT) were used to evaluate albendazole efficacy in three different areas of South Darfur using naturally (Rehed Al-Birdi and Tulus) and experimentally infected (Tulus and Um Dafuq) goats. Using samples from Central, East and South Darfur, pyro- and Sanger sequencing were used to detect the polymorphisms F167Y, E198A and F200Y in H. contortus isotype 1 ß-tubulin in DNA extracted from pooled third-stage larval (L3) samples (n = 36) on days 0 and 10 during trials, and from pooled adult male H. contortus (treated goats, n = 14; abattoirs, n = 83) including samples from populations previously found to be resistant in South Darfur. RESULTS: Albendazole efficacies at 5, 7.5 and 10 mg/kg doses were 73.5-90.2% on day 14 in natural and experimental infections while 12.5 mg/kg showed > 96.6% efficacy. EC50 in the EHT were 0.8 and 0.11 µg/ml thiabendazole in natural and experimental infection trials, respectively. PCRs detected Haemonchus, Trichostrongylus and Cooperia in L3 samples from albendazole-treated goats. Haemonchus contortus allele frequencies in codons 167 and 200 using pyrosequencing assays were ≤ 7.4% while codon 198 assays failed. Sanger sequencing revealed five novel polymorphisms at codon 198. Noteworthy, an E198L substitution was present in 82% of the samples (L3 and adults) including all post-treatment samples. Moreover, E198V, E198K and potentially E198I, and E198Stop were identified in a few samples. CONCLUSIONS: To our knowledge, this is the first report of E198L in BZ resistant H. contortus and the second where this is the predominant genotype associated with resistance in any strongyle species. Since this variant cannot be quantified using pyrosequencing, the results highlight important limitations in the general applicability of pyrosequencing to quantify BZ resistance genotypes.


Assuntos
Benzimidazóis/farmacologia , Códon , Resistência a Medicamentos/genética , Doenças das Cabras/parasitologia , Haemonchus/genética , Polimorfismo de Nucleotídeo Único , Tubulina (Proteína)/genética , Albendazol/farmacologia , Animais , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Feminino , Frequência do Gene , Genótipo , Doenças das Cabras/tratamento farmacológico , Cabras , Haemonchus/efeitos dos fármacos , Masculino , Análise de Sequência de DNA , Sudão , Trichostrongyloidea/genética , Trichostrongylus/genética
16.
J Agric Food Chem ; 68(14): 4176-4186, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32181655

RESUMO

Worldwide, parasitic gastrointestinal nematodes continue to threaten animal health, welfare, and production in outdoor breeding systems of small ruminants. For more than 50 years, the control of these parasitic worms has relied on the use of commercial synthetic anthelmintics. However, anthelmintic resistance in worm populations is nowadays widespread and requires novel solutions. The use of tannin-rich plants has been suggested as an alternative to synthetic anthelmintics to control gastrointestinal nematodes. The majority of previous studies have focused on the activity of proanthocyanidins (syn condensed tannins), and less is known about ellagitannins. In this study, the effects of 30 structurally unique ellagitannins on the exsheathment of third-stage infective larvae were examined on Haemonchus contortus and Trichostrongylus colubriformis by the in vitro larval exsheathment inhibition assay. Ellagitannins were found to be promising natural anthelmintics as they showed direct inhibition on larval exsheathment for both nematode species. In general, ellagitannins were more efficient at inhibiting the exsheathment of H. contortus larvae than those of T. colubriformis. The efficiency of inhibition increased as the degree of oligomerization or the molecular weight of the ellagitannin increased. Otherwise, we found no other structural features of ellagitannins that significantly affected the anthelmintic activity on the third-stage infective larvae. The effective concentrations were physiologically relevant and should be achievable in the gastrointestinal tract also in in vivo conditions.


Assuntos
Haemonchus/efeitos dos fármacos , Taninos Hidrolisáveis/química , Larva/efeitos dos fármacos , Extratos Vegetais/química , Tricostrongiloidíase/tratamento farmacológico , Trichostrongylus/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Resistência a Medicamentos , Haemonchus/parasitologia , Taninos Hidrolisáveis/farmacologia , Larva/parasitologia , Estrutura Molecular , Peso Molecular , Extratos Vegetais/farmacologia , Polifenóis/química , Relação Estrutura-Atividade , Trichostrongylus/parasitologia
17.
Vet Parasitol ; 279: 109010, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32035291

RESUMO

In a context of nematodicidal resistance, anthelmintic combinations have emerged as a reliable pharmacological strategy to control gastrointestinal nematodes in grazing systems of livestock production. The current work evaluated the potential drug-drug interactions following the coadministration of two macrocyclic lactones (ML) ivermectin (IVM) and abamectin (ABM) to parasitized cattle using a pharmacokinetic/pharmacodynamic (PK/PD) approach. The kinetic behavior of both compounds administered either separately or coadministered was assessed and the therapeutic response of the combination was evaluated under different resistance scenarios. In the pharmacological trial, calves received a single subcutaneous (s.c.) injection of IVM (100 µg/Kg); a single s.c. injection of ABM (100 µg/Kg) or IVM + ABM (50 µg/Kg each) administered in different injection sites to reach a final ML dose of 100 µg/Kg (Farm 1). Plasma samples were taken from those animals up to 20 days post-treatment. IVM and ABM plasma concentrations were quantified by HPLC. A parasitological trial was carried out in three farms with different status of nematodes resistance to IVM. Experimental animals received IVM (200 µg/Kg), ABM (200 µg/Kg) or IVM + ABM (100 µg/Kg each) in Farm 2, and IVM + ABM (200 µg/Kg each) in Farms 3 and 4. The anthelmintic efficacy was determined by fecal egg count reduction test (FECRT). PK analysis showed similar trends for IVM kinetic behavior after coadministration with ABM. Conversely, the ABM elimination half-life was prolonged and the systemic exposure during the elimination phase was increased in the presence of IVM. Although IVM alone failed to control Cooperia spp., the combination IVM + ABM was the only treatment that achieved an efficacy higher than 95% against resistant Cooperia spp. in all farms. In fact, when Cooperia spp. was the main genus within the nematode population and Haemonchus spp. was susceptible or slightly resistant to ML (Farms 2 and 4), the total FECR for the combination IVM + ABM was higher than 90%. Instead, when the predominant nematode genus was a highly resistant Haemonchus spp. (Farm 3), the total FECR after the combined treatment was as low as the single treatments. Therefore, the rational use of these pharmacological tools should be mainly based on the knowledge of the epidemiology and the nematode susceptibility status in each cattle farm.


Assuntos
Antinematódeos/farmacologia , Doenças dos Bovinos/tratamento farmacológico , Haemonchus/efeitos dos fármacos , Ivermectina/análogos & derivados , Ivermectina/farmacologia , Rabditídios/efeitos dos fármacos , Animais , Antinematódeos/farmacocinética , Bovinos , Interações Medicamentosas , Hemoncose/tratamento farmacológico , Hemoncose/veterinária , Ivermectina/farmacocinética , Masculino , Distribuição Aleatória , Infecções por Rhabditida/tratamento farmacológico , Infecções por Rhabditida/veterinária
18.
Parasite ; 27: 3, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31934848

RESUMO

Eukaryote plasma membranes protect cells from chemical attack. Xenobiotics, taken up through passive diffusion, accumulate in the membranes, where they are captured by transporters, among which P-glycoproteins (Pgps). In nematodes such as Haemonchus contortus, eggshells and cuticles provide additional protective barriers against xenobiotics. Little is known about the role of these structures in the transport of chemical molecules. Pgps, members of the ABC transporter family, are present in eggshells and cuticles. Changes in the activity of these proteins have also been correlated with alterations in lipids, such as cholesterol content, in eggshells. However, the cellular mechanisms underlying these effects remain unclear. We show here that an experimental decrease in the cholesterol content of eggshells of Haemonchus contortus, with Methyl-beta-CycloDextrin (MßCD), results in an increase in membrane fluidity, favouring Pgp activity and leading to an increase in resistance to anthelmintics. This effect is modulated by the initial degree of anthelminthic resistance of the eggs. These results suggest that eggshell fluidity plays a major role in the modulation of Pgp activity. They confirm that Pgp activity is highly influenced by the local microenvironment, in particular sterols, as observed in some vertebrate models. Thus, eggshell barriers could play an active role in the transport of xenobiotics.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Exoesqueleto/química , Membrana Celular/química , Colesterol/química , Resistência a Medicamentos , Haemonchus/química , Haemonchus/efeitos dos fármacos , Animais , Anti-Helmínticos/farmacologia , Membrana Celular/efeitos dos fármacos , Fluidez de Membrana , Xenobióticos/farmacologia
19.
Parasitol Int ; 76: 102063, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31978599

RESUMO

Pour-on eprinomectin was recently registered for lactating small ruminants. Given the high prevalence of benzimidazole resistance in gastrointestinal nematodes in dairy goats, many farmers use eprinomectin exclusively to treat their animals. On a French dairy goat farm, a veterinary practitioner noted a poor response to two types of eprinomectin treatment (pour-on application and injectable formulation). Therefore, we evaluated the efficacy of both formulations of eprinomectin, as well as moxidectin and fenbendazole, using the fecal egg count reduction test (FECRT) according to the World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. Nematode species were identified at days 0 and post-treatment days 14 after bulk larval cultures, by morphology and real-time PCR. Plasma concentrations of eprinomectin were analyzed by high-performance liquid chromatography (HPLC) at post-treatment days 2 and 5 in the eprinomectin-treated groups. Egg count reductions were poor in animals treated with topical (-16.7%; 95% CI:[-237; 59]) or subcutaneous (21.5%; 95% CI:[-126; 73]) eprinomectin, and with fenbendazole (-5.8%; 95% CI:[-205; 63]). Haemonchus contortus was the main species identified by morphology and by real-time PCR before and after treatment. The plasma concentrations of eprinomectin were determined in all eprinomectin-treated animals and were above 2 ng/ml at post-treatment day 2, indicating that the lack of effect was not due to low exposure of the worms to the drug. Interestingly, moxidectin remained effective in all infected animals. This is the first report of multiple resistance to eprinomectin and benzimidazole in H. contortus on a French dairy goat farm with moxidectin as a relevant alternative.


Assuntos
Anti-Helmínticos/uso terapêutico , Benzimidazóis/uso terapêutico , Resistência a Múltiplos Medicamentos , Cabras/parasitologia , Hemoncose/veterinária , Haemonchus/efeitos dos fármacos , Ivermectina/análogos & derivados , Animais , Anti-Helmínticos/sangue , Benzimidazóis/sangue , Fazendas , Feminino , França , Doenças das Cabras/tratamento farmacológico , Doenças das Cabras/parasitologia , Hemoncose/tratamento farmacológico , Ivermectina/sangue , Ivermectina/uso terapêutico , Contagem de Ovos de Parasitas
20.
Acta Trop ; 201: 105213, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31600523

RESUMO

The aim of the present study was to evaluate the in vitro effect of quercetin combined with ivermectin (IVM) on Haemonchus contortus larvae and adults with different resistance profiles and demonstrate the in vivo anthelmintic action of this combination when used in sheep naturally infected. The effect of combination was evaluated based on the analysis of the mean effective concentration (EC50) obtained for larvae using the larval migration inhibition test and for adults using the motility test on females. The tests with larvae and adults were conducted using isolates with different degrees of susceptibility to IVM (sensitive, intermediate and highly resistant). The in vivo effect was evaluated based on the reduction in the egg count (FEC) and reduction in the count of adult helminths recovered after parasitological necropsy. Using the combination of quercetin with IVM, it was observed that in larvae, quercetin did not significantly reduce the EC50 for IVM in the sensitive and highly resistant isolates, but led to a significant reduction in the EC50 for IVM in the intermediate isolate. In adults, quercetin did not significantly reduce the EC50 for IVM in any of the isolates. No significant effect of the combination was found regarding the reduction in FEC or total count of parasites. The results of the in vitro and in vivo tests performed in the present study on quercetin activity underscore the importance of evaluating resistance-reversing agents among different stages of parasite development as well as among isolates with different resistance profiles. The action of quercetin combined with IVM on the motility of H. contortus larvae and adults was influenced by the degree of resistance and development stage of the parasite. The combination was effective only on intermediate resistant larvae. No action of the combination against adults was found. Moreover, this combination, when administered through the intra-abomasal route, was not effective at reducing the FEC and parasite load of naturally infected sheep.


Assuntos
Anti-Helmínticos/uso terapêutico , Resistência a Medicamentos/efeitos dos fármacos , Hemoncose/tratamento farmacológico , Haemonchus/efeitos dos fármacos , Ivermectina/uso terapêutico , Larva/efeitos dos fármacos , Quercetina/uso terapêutico , Doenças dos Ovinos/tratamento farmacológico , Animais , Feminino , Masculino , Contagem de Ovos de Parasitas , Ovinos/parasitologia , Doenças dos Ovinos/parasitologia
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