Characterization of the eggshell of Haemonchus Contortus—I. Structural components

Thymol is a monoterpene and acetylation form of this compound can reduce the toxicity and enhance its biological effects. The objective of this study was to evaluate the effect of thymol and thymol acetate (TA) on egg, larva and adult Haemonchus contortus and the cuticular changes, acute toxicity in mice and the efficacy on sheep gastrointestinal nematodes. In vitro tests results were analyzed by analysis of variance (ANOVA) and followed by comparison with Tukey test or Bonferroni. The efficacy of in vivo test was calculated by the BootStreet program. In the egg hatch test (EHT), thymol (0.5 mg/mL) and TA (4 mg/mL) inhibited larval hatching by 98% and 67.1%, respectively. Thymol and TA (8 mg/mL) inhibited 100% of larval development. Thymol and TA (800 µg/mL) reduced the motility of adult worms, by 100% and 83.4%, respectively. Thymol caused cuticular changes in adult worm teguments. In the acute toxicity test, the LD 50 of thymol and TA were 1,350.9 mg/kg and 4,144.4 mg/kg, respectively. Thymol and TA reduced sheep egg count per gram of faeces (epg) by 59.8% and 76.2%, respectively. In in vitro tests thymol presented better anthelmintic activity than TA. However TA was less toxic and in in vivo test efficacy was similar.Keywords: Acetylation, thymol, Haemonchus contortus, nematodes, sheep. ResumoTimol é um monoterpeno e a acetilação deste composto pode reduzir a toxicidade e potencializar os seus efeitos biológicos. O objetivo deste trabalho foi avaliar o efeito do timol e acetato de timolila (AT) sobre ovos, larvas e adultos de Haemonchus contortus e suas alterações cuticulares, toxicidade aguda em camundongos e a eficácia sobre nematoides gastrintestinais de ovinos. Os resultados dos testes in vitro foram analisados por análise de variância (ANOVA) e comparados pelo testes de Tukey ou Bonferroni. A eficácia do teste de redução da contagem de ovos nas fezes (RCOF) foi calculada pelo programa BootStreet. No teste de inibição da eclosão de ovos (TEO), timol (0,5 mg/mL) e AT (4 mg/mL) inibiram a eclosão das larvas em 98% e 67,1%, respectivamente. Timol e AT (8 mg/mL) inibiram 100% do desenvolvimento larval. Timol e AT (800 µg/mL) reduziram a motilidade dos nematoides adultos, em 100% e 83,4%, respectivamente. O timol provocou alterações cuticulares nos nematoides adultos. No teste de toxicidade aguda, a DL50 do timol e AT foi de 1.350,9 mg/kg e 4.144,4 mg/kg, respectivamente. Timol e AT reduziram a contagem de ovos por gramas de fezes (OPG) dos ovinos em 59,8% e 76,2%, respectivamente. Nos testes in vitro timol apresentou atividade melhor anti-helmíntica do que AT. Entretanto, AT foi menos tóxico do que o timol e no teste in vivo apresentaram eficácia semelhante.

Section: Discussionmentioning

confidence: 97%

Anthelmintic effect of thymol and thymol acetate on sheep gastrointestinal nematodes and their toxicity in mice

André

Cavalcante

Ribeiro

et al. 2017

“…In nematodes, metalloproteinase enzymes play important roles in the formation of the cuticle, and protease inhibitors can thus be used to inhibit and prevent the development of the parasite by interfering with these enzymes (LAWRENCE & KOUNDAL, 2002). Moreover, the shells of H. contortus eggs have a protein layer that includes several proteases (MANSFIELD et al, 1992). Because proteases are present in all stages of nematode development, the use of protease inhibitors may represent a highly effective means of controlling these parasites.…”

Section: Discussionmentioning

confidence: 99%

“…Chitin is an important component of egg shells, and acts as a protective barrier against the external environment ( ROGERS & BROOKS, 1977). The main proteins of the cuticle of H. contortus eggs are degraded by the enzymes proteinase K and chitinase (MANSFIELD et al, 1992). Pathogenic fungi and some nematodes use chitinase to break through this protective barrier and penetrate the host (LEGER et al, 1993;HUBER et al, 1991;SHAHABUDDIN et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

“…The cuticle of H. contortus eggs has three layers: an internal layer composed of lipids with some associated proteins; an intermediate layer composed of chitin fibers surrounded by proteins; and an outer layer composed of proteins and lipids (MANSFIELD et al, 1992). Different classes of proteases are known to be inactivated by plant proteins, called protease inhibitors (OLIVA et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Anthelmintic activity of Leucaena leucocephala protein extracts on Haemonchus contortus

Soares

Araújo

Lopes

et al. 2015

The objective of this study was to evaluate the effects of protein extracts obtained from the plant Leucaena leucocephala on the nematode parasite Haemonchus contortus. The seeds, shell and cotyledon of L. leucocephala were separated and their proteins extracted using a sodium phosphate buffer, and named as TE (total seed extract), SE (shell extract) and CE (cotyledon extract). Soluble protein content, protease, protease inhibitory and chitinase activity assays were performed. Exsheathment inhibition of H. contortus larvae were performed at concentrations of 0.6 mg mL . The effective concentration for 50% hatching inhibition (EC 50 ) was estimated by probit. Different proportions of soluble proteins, protease and chitinase were found in TE and CE. Protease inhibitory activity was detected in all extracts. The EC 50 of the CE and TE extracts were 0.48 and 0.33 mg mL -1 , respectively. No ovicidal effects on H. contortus were detected in SE extracts, and none of the protein extracts demonstrated larvicidal effects on H. contortus. We therefore conclude that protein extracts of L. leucocephala had a detrimental effect on nematode eggs, which can be correlated with the high protease and chitinase activity of these extracts.Keywords: Plant proteins, protease, protease inhibitor, chitinase. ResumoO objetivo deste estudo foi avaliar a atividade anti-helmíntica de extratos proteicos de leucena (Leucaena leucocephala) sobre Haemonchus contortus. As sementes, as cascas e os cotilédones foram moídos separadamente e as proteínas extraídas com tampão fosfato de sódio e denominados: TE (extrato total), SE (extrato casca) e CE (extrato cotilédone). O teor de proteínas, atividade proteolítica, inibitória de protease e quitinolítica dos extratos foram verificados, além da ação sobre a eclosão de ovos e desembainhamento larvar de H. contortus. A concentração efetiva para inibição de 50% da eclosão dos ovos (EC 50 ) foi calculada através do probit. Foi demonstrado que TE e CE possuem, em diferentes proporções, proteínas solúveis, protease e quitinase. Atividade inibitória de protease foi encontrada em todos os extratos. A EC 50 dos extratos CE e TE foram 0,48 e 0,33 mg de proteína mL -1 , respectivamente. O extrato SE não apresentou atividade sobre a eclosão dos ovos. Os extratos proteicos não apresentaram efeito larvicida sobre H. contortus. Conclui-se que a ação de extratos proteicos de L. leucocephala afetam negativamente a eclodibilidade dos ovos, correlacionando-se com a alta atividade de protease e quitinase dos extratos testados.Palavras-chaves: Proteínas vegetais, protease, inibidor de protease, quitinase.

“…The eggshell of H. contortus consists of three basic layers containing proteins , an outer vitelline layer, a middle chitinous layer and an inner lipid layer, that are digested during hatching (MANSFIELD et al, 1992). In addition, like embryonic development, proteolysis is an essential metabolic process required for protein processing.…”

Section: Introductionmentioning

confidence: 99%

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus

Salles

Braga

Nascimento³

et al. 2014

Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (<12 kDa) for Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P<0.05, Bonferroni test). The two fractions of Crotalaria spectabilis showed the same ovicidal activity (P>0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.Keywords: Goats, nematodes, egg hatch assay, lectin, protease inhibitors, hemolysin. ResumoMoléculas bioativas de espécies vegetais são alternativas promissoras ao controle químico dos nematoides gastrintestinais em ruminantes. Extratos de sementes de espécies nativas e exóticas do Semiárido Brasileiro foram testados in vitro em ensaio de eclosão de ovos e investigada a natureza proteica da bioatividade. Três extrações com três solventes foram feitas para cada semente estudada. Todas as sementes apresentaram atividade ovicida, variando com o solvente utilizado. Maior taxa de inibição da eclosão concentrou-se nas frações de moléculas de baixa massa molecular (<12 kDa) para Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera e Ricinus communis (P<0,05, teste de Bonferroni). Crotalaria spectabilis mostrou atividade nas duas frações, sem diferença entre elas (P>0,05, teste de Bonferroni). Observou-se atividade hemaglutinante nas frações de C. spectabilis e M. oleifera, de hemolisina em A. lebbeck e M. oleifera, de atividade inibidora de protease da serina em A. lebbeck, I. asarifolia, J. curcas, M. oleifera