RESUMO
The excretory/secretory (E/S) products released by infective transitory larvae (xL3) of Haemonchus placei have an important biological function in stimulating immune mechanisms during the invasive process. Our objective was to analyse the modulatory activity of 15 and 70 kDa E/S products from H. placei xL3. Both E/S products were collected from xL3in vitro cultures at 24 and 72 h. Proteins were confirmed by SDS-PAGE, and the corresponding spots were elicited by gel isoelectrofocusing (IEF) and characterised by mass spectrometry. Additionally, flow cytometry of CD4+/γδ+ T cells and immune gene expression were performed by proliferation assays using each E/S product to stimulate lymphocyte and peripheral blood mononuclear cells (PBMCs) from non-infected calves. The IEF results displayed two spots of 7.0 and 5.7 pI for the 15 and 70 kDa products, respectively. Additionally, 29 and 17 peptides from the 15 and 70 kDa E/S products, respectively, were identified with the hypothetical neurotransmitter and enzymatic functions necessary for larval development. The relative expression displayed upregulation of IL4, 5, 6, 8, 10, 13, IFNγ, and FCεR1A genes (from 2.0- to 17.6-fold, p < 0.05) stimulated by the 15 and 70 kDa proteins, indicating specific genes against haemonchosis. Although the percentage of median florescence intensity (MFI%) of CD4+/γδ+ T cells did not change for both E/S products compared to the negative control and concanavalin-A stimulated cells as the positive control (p > 0.05), the 15-kDa protein reduced the levels of both T cells, and the 70-kDa proteins increased the γδ+ cells slightly. Additionally, there was increased PBMCs proliferation by the 70 kDa proteins (p < 0.05), denoting the biological role of other immune cells. The 15 and 70 kDa protein E/S products from H. placei xL3 showed modulation of the immune response, and although more studies are required, they indicate important functions in the host/parasite interaction.
Assuntos
Antígenos de Helmintos , Haemonchus , Larva , Leucócitos Mononucleares , Linfócitos , Animais , Antígenos de Helmintos/farmacologia , Bovinos , Haemonchus/química , Interações Hospedeiro-Parasita/imunologia , Fatores Imunológicos/farmacologia , Larva/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologiaRESUMO
This study aimed to evaluate the anthelmintic and ultrastructural effects of Calotropis procera latex on Haemonchus contortus. C. procera latex was twice centrifuged at 10,000×g and dialyzed to obtain a fraction rich in proteins, named LP (latex protein), and at 3,000 rpm to obtain a fraction rich in secondary metabolites, named LNP (latex non-protein). Specimens of H. contortus exposed to LNP, LP and PBS in the Adult Worm Motility Test (AWMT) were submitted to scanning (SEM) and transmission (TEM) electron microscopy to verify changes in their ultrastructure. Phytochemical tests in the LNP indicated the presence of phenols, steroids, alkaloids and cardenolides. High-Performance Liquid Chromatography (HPLC) characterized the presence of the compounds gallic acid and quercetin in the LNP. The protein content in the LP was 43.1 ± 1.1 mg/mL and 7.7 ± 0.3 mg/mL in LNP. In AWMT, LNP and LP inhibited the motility of 100% of the nematodes, with LNP being more effective than LP and ivermectin more effective than both (p <0.05). Cuticle changes were observed by SEM and TEM in nematodes treated with LP and LNP. Calotropis procera latex has anthelmintic effects against H. contortus, causing damage to its cuticle and other alterations in its ultrastructure.(AU)
Este estudo objetivou avaliar os efeitos anti-helmínticos e ultraestruturais do látex de Calotropis procera sobre Haemonchus contortus. Látex de C. procera foi centrifugado duas vezes à a 10.000xg e dialisado para obter uma fração rica em proteínas, denominada proteínas do látex (LP). E centrifugado e centrifugado a 3.000 rpm, para obter uma fração rica em metabólitos secundários, denominada LNP (látex não proteico). Espécimes de H. contortus expostos à LNP, LP e PBS no Teste de Motilidade dos Nematoides Adultos (TMNA) foram submetidos a microscopia eletrônica de varredura (MEV) e de transmissão (MET), para verificar alterações em sua ultraestrutura. Testes fitoquímicos em LNP indicaram a presença de fenóis, esteroides, alcaloides e cardenolídeos. A presença dos compostos ácido gálico e quercetina em LNP foi caracterizada por Cromatografia Líquida de Alta Eficiência (CLAE). O conteúdo de proteínas em LP foi de 43,1 ± 1,1 mg/mL e de 7,7 ± 0,3 mg/mL em LNP. No TMNA, LNP e LP inibiram a motilidade de 100% dos nematoides, sendo LNP mais eficaz que LP, e a ivermectina mais eficaz que ambos (p <0,05). Alterações na cutícula de nematoides tratados com LP e LNP foram observadas por MEV e MET. O látex de C. procera apresenta efeito anti-helmíntico sobre H. contortus, causando danos à sua cutícula e outras alterações em sua ultraestrutura.(AU)
Assuntos
Animais , Calotropis/química , Haemonchus/química , Haemonchus/parasitologia , Anti-HelmínticosRESUMO
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.
Assuntos
Cromatografia de Afinidade/métodos , Haemonchus/química , Mesocestoides/química , Proteínas Associadas aos Microtúbulos/metabolismo , Tubulina (Proteína)/isolamento & purificação , Sequência de Aminoácidos , Animais , Infecções por Cestoides/parasitologia , Escherichia coli/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Hemoncose/parasitologia , Hemoncose/veterinária , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ovinos , Doenças dos Ovinos/parasitologia , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Suínos , Tubulina (Proteína)/química , Tubulina (Proteína)/genéticaRESUMO
This study aimed to evaluate the in vitro anthelmintic activity of Mimosa caesalpinifolia (sabiá) ketone extract, which is rich in condensed tannins (CT), on Haemonchus contortus and Trichostrongylus colubriformis. The leaves and stems of M. caesalpinifolia were collected, dried in the shade, and ground to a dry powder from which ketone extracts were obtained for in vitro assays. The extracts were diluted in 5% acetone and PBS to concentrations in the range 0.25-2.80 mg mL-¹ and the inhibition of egg hatchability was assayed. Feces collected from goats naturally infected with H. contortus (55%) and T. colubriformis (45%) were macerated and the helminth eggs were collected to conduct in vitro egg hatchability tests. Nematode larvae collected from the feces were used for a larval exsheathment inhibition assay of M. caesalpinifolia leaf and M. caesalpinifolia leaf and stem ketone extracts at concentrations of 0.6 and 1.2 mg mL-¹; three replicates were performed for each concentration. PBS was used as a negative control. Polyvinylpolypyrrolidone (PVPP) was added to the extracts to assess the effect of phenolic compounds on anthelmintic activity. An inhibitory effect of 80.7% and 82.3% was observed on hatchability after treatment with 1.0 and 2.0 mg mL-¹ M. caesalpinifolia leaf ketone extract, respectively. An inhibition rate lower than 75% was observed for 1.0 mg mL-¹ M. caesalpinifolia leafand stem ketone extract. The M. caesalpinifolia leaf ketone extract larval exsheathment assay resultedin inhibition of 97.3% and 99.8% at concentrations of 0.6 and 1.2 mg mL-¹, respectively. For the leafand stem ketone extract, the larval exsheathment inhibition was 94.3% at 1.2 mg mL-¹. The leaf andleaf and stem extracts showed no inhibitory effects on larval exsheathment after the addition of PVPP.These results showed that cuticular loss was related to the action of the phenolic compounds in the M.caesalpinifolia extracts. M. caesalpinifolia leaf ketone...(AU)
Este trabalho teve como objetivo avaliar a atividade anti-helmíntica in vitro do extrato cetônico, rico em Tanino Condensado (TC) de Mimosa caesalpinifolia (sabiá) sobre Haemonchus contortus e Trichostrongylus colubriformis. Foram coletadas folhas e caules, secas a sombra por sete dias e moídas. O pó seco das folhas e caules foi utilizado para obtenção dos extratos cetônicos e realização dos testes in vitro. Os extratos foram diluídos em acetona 5% e PBS em concentrações entre 0,25 e 2,80 mg mL-¹ para realização do teste de inibição da eclodibilidade de ovos. Fezes coletadas de caprino infectado com H. contortus (55%) e T. colubriformis (45%) foram maceradas e os ovos dos helmintos recuperados, e testes in vitro de eclodibilidade dos ovos realizados. Larvas dos nematódeos recuperadas das fezes foram utilizadas para o teste de inibição do desembainhamento larvar com os extratos de folhas, e folhas com caule de M. caesalpinifolia em concentrações de 0,6 a 1,2 mg mL-¹ com três repetições para cada concentração. O controle negativo foi realizado com PBS. Polivinilpolipirrolidona (PVPP) foi adicionado aos extratos para verificar o efeito de compostos fenólicos na atividade anti-helmíntica. No extrato cetônico de folha de M. caesalpinifolia, nas concentrações 1,0 e 2,0 mg mL-¹, foi observado uma taxa de inibição de eclodibilidade dos ovos de 80,7 e 82,3%, respectivamente. O extrato cetônico da folha de M. caesalpinifolia inibiu a eclodibilidade dos ovos,entretanto o extrato da folha e caule apresentou eficiência inferior, demonstrando que os compostosativos estão mais presentes nas folhas. Para a inibição do desembainhamento larvar os extratos cetônicosda M. caesalpinifolia foram eficazes na maior concentração testada. Com esses resultados, pesquisasin vivo devem ser realizadas para comprovação da atividade anti-helmíntica dessa planta em caprinos.(AU)
Assuntos
Animais , Trichostrongylus/química , Haemonchus/química , Mimosa/química , Corpos Cetônicos/análise , Corpos Cetônicos/química , RuminantesRESUMO
This study aimed to evaluate the in vitro anthelmintic activity of Mimosa caesalpinifolia (sabiá) ketone extract, which is rich in condensed tannins (CT), on Haemonchus contortus and Trichostrongylus colubriformis. The leaves and stems of M. caesalpinifolia were collected, dried in the shade, and ground to a dry powder from which ketone extracts were obtained for in vitro assays. The extracts were diluted in 5% acetone and PBS to concentrations in the range 0.25-2.80 mg mL-¹ and the inhibition of egg hatchability was assayed. Feces collected from goats naturally infected with H. contortus (55%) and T. colubriformis (45%) were macerated and the helminth eggs were collected to conduct in vitro egg hatchability tests. Nematode larvae collected from the feces were used for a larval exsheathment inhibition assay of M. caesalpinifolia leaf and M. caesalpinifolia leaf and stem ketone extracts at concentrations of 0.6 and 1.2 mg mL-¹; three replicates were performed for each concentration. PBS was used as a negative control. Polyvinylpolypyrrolidone (PVPP) was added to the extracts to assess the effect of phenolic compounds on anthelmintic activity. An inhibitory effect of 80.7% and 82.3% was observed on hatchability after treatment with 1.0 and 2.0 mg mL-¹ M. caesalpinifolia leaf ketone extract, respectively. An inhibition rate lower than 75% was observed for 1.0 mg mL-¹ M. caesalpinifolia leafand stem ketone extract. The M. caesalpinifolia leaf ketone extract larval exsheathment assay resultedin inhibition of 97.3% and 99.8% at concentrations of 0.6 and 1.2 mg mL-¹, respectively. For the leafand stem ketone extract, the larval exsheathment inhibition was 94.3% at 1.2 mg mL-¹. The leaf andleaf and stem extracts showed no inhibitory effects on larval exsheathment after the addition of PVPP.These results showed that cuticular loss was related to the action of the phenolic compounds in the M.caesalpinifolia extracts. M. caesalpinifolia leaf ketone...
Este trabalho teve como objetivo avaliar a atividade anti-helmíntica in vitro do extrato cetônico, rico em Tanino Condensado (TC) de Mimosa caesalpinifolia (sabiá) sobre Haemonchus contortus e Trichostrongylus colubriformis. Foram coletadas folhas e caules, secas a sombra por sete dias e moídas. O pó seco das folhas e caules foi utilizado para obtenção dos extratos cetônicos e realização dos testes in vitro. Os extratos foram diluídos em acetona 5% e PBS em concentrações entre 0,25 e 2,80 mg mL-¹ para realização do teste de inibição da eclodibilidade de ovos. Fezes coletadas de caprino infectado com H. contortus (55%) e T. colubriformis (45%) foram maceradas e os ovos dos helmintos recuperados, e testes in vitro de eclodibilidade dos ovos realizados. Larvas dos nematódeos recuperadas das fezes foram utilizadas para o teste de inibição do desembainhamento larvar com os extratos de folhas, e folhas com caule de M. caesalpinifolia em concentrações de 0,6 a 1,2 mg mL-¹ com três repetições para cada concentração. O controle negativo foi realizado com PBS. Polivinilpolipirrolidona (PVPP) foi adicionado aos extratos para verificar o efeito de compostos fenólicos na atividade anti-helmíntica. No extrato cetônico de folha de M. caesalpinifolia, nas concentrações 1,0 e 2,0 mg mL-¹, foi observado uma taxa de inibição de eclodibilidade dos ovos de 80,7 e 82,3%, respectivamente. O extrato cetônico da folha de M. caesalpinifolia inibiu a eclodibilidade dos ovos,entretanto o extrato da folha e caule apresentou eficiência inferior, demonstrando que os compostosativos estão mais presentes nas folhas. Para a inibição do desembainhamento larvar os extratos cetônicosda M. caesalpinifolia foram eficazes na maior concentração testada. Com esses resultados, pesquisasin vivo devem ser realizadas para comprovação da atividade anti-helmíntica dessa planta em caprinos.
Assuntos
Animais , Corpos Cetônicos/análise , Corpos Cetônicos/química , Haemonchus/química , Mimosa/química , Ruminantes , Trichostrongylus/químicaRESUMO
A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in four groups of nine worm-free calves challenged with either 8000 H. contortus or Haemonchus placei infective larvae. Vaccinates received 50 µg of the antigen and 1 mg QuilA adjuvant three times 21 days apart, while the controls got adjuvant alone. The calves were challenged 7 days after the last immunization and killed for worm counts 43 days later. Immunization resulted in high titre antibodies against the vaccine antigens and significant reduction in egg output and worm numbers of both challenge species, compared with control calves. It was concluded that vaccination of calves with native parasite gut membrane glycoproteins obtained from H. contortus conferred protection against both H. placei and H. contortus.
Assuntos
Antígenos de Helmintos/imunologia , Hemoncose/prevenção & controle , Haemonchus/imunologia , Imunização/métodos , Proteínas de Membrana/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/isolamento & purificação , Bovinos , Doenças dos Bovinos/imunologia , Doenças dos Bovinos/prevenção & controle , Modelos Animais de Doenças , Trato Gastrointestinal/química , Trato Gastrointestinal/imunologia , Hemoncose/imunologia , Haemonchus/química , Proteínas de Membrana/isolamento & purificação , Contagem de Ovos de Parasitas , Saponinas de Quilaia , Saponinas/administração & dosagemRESUMO
Control of Haemonchus placei, one of the most important cattle nematodes in Brazil, relies on the use of anthelmintics. However, there is a need for integrated control, which includes active immunization. The aim of this work was to assess the protection afforded to calves by immunization with adult H. placei extracts against a high-dose challenge infection, a condition frequently found in the tropics. Holstein calves aged 8-10 months were immunized four times with intestinal extracts (Group D) or with a Triton X-100-soluble fraction of adult H. placei (Group A), challenge-infected with 120,000 infective larvae and sacrificed 40 days later. Immunized animals had higher IgG titers than the controls against tested fractions after the 2nd immunization, peaking after the 4th. Sera from both immunized groups recognized bands of similar apparent mass in both antigenic preparations, some of which were similar in molecular weight to Haemonchus contortus antigens with known protective effect to sheep. Egg counts were 49% and 57% lower in Groups A and D than in controls, respectively. High levels of protection were observed in two of the four calves in Group D, as evidenced by very low worm numbers recovered at necropsy, absence of eggs in the uteri of the recovered females and reduced worm length. Group D animals also showed milder signs of anemia than the other infected animals. Results demonstrate that protection against homologous high-dose challenge can be achieved by immunizing calves with H. placei gut antigens.