Phenotypic and genotypic approaches for detection of anthelmintic resistant sheep gastrointestinal nematodes from Brazilian northeast.

The aim of this study was to characterize the anthelmintic resistance (AR) of a sheep gastrointestinal nematode population, named Caucaia, from northeastern Brazil. Phenotypic tests performed were: egg hatch (EHT), larval development (LDT) and fecal egg count reduction (FECRT). Benzimidazoles (BZs) genotypic evaluation was by frequency of single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A, and for levamisole (LEV), by frequency of resistance alleles of Hco-acr-8 gene. The primers were designed specifically for Haemonchus contortus. Effective concentrations 50% (EC50) for BZs (EHT), and for macrocyclic lactones (MLs) and LEV (LDT) were 1.02 µg/mL, 1.81 ng/mL and 0.04 µg/mL, respectively. Resistance ratios for MLs and LEV were 0.91 and 3.07, respectively. FECRT efficacies of BZs, MLs, monepantel (MPTL) and LEV were 52.4; 87.0; 94.5 and 99.6%, respectively. qPCR for BZs demonstrated resistance allele frequencies of 0%, 26.24% and 69.08% for SNPs E198A, F200Y and F167Y, respectively. For LEV, 54.37% of resistance alleles were found. There was agreement between EHT, FECRT and qPCR for BZs, and agreement between LDT and qPCR for LEV. Thus, based on higher sensitivity of qPCR, and phenotypic evaluation, the Caucaia population was considered resistant to BZs, MLs, LEV and suspect for MPTL.

Anthelmintic activity of Eucalyptus citriodora essential oil and its major component, citronellal, on sheep gastrointestinal nematodes.

This study aimed to evaluate the anthelmintic activity of Eucalyptus citriodora essential oil and citronellal on sheep gastrointestinal nematodes. Essential oil composition was determined by gas chromatography mass spectrometry. The substances were evaluated in vitro using adult worm motility test (AWMT) and transmission electron microscopy (TEM). The acute toxicity test in mice and the fecal egg count reduction test (FECRT) in sheep were performed. Citronellal was confirmed as the essential oil major constituent (63.9%). According to the AWMT, 2 mg/mL of essential oil and citronellal completely inhibited Haemonchus contortus motility at 6 h post exposure. H. contortus exposed to essential oil and citronellal exhibited internal ultrastructural modifications. The lethal dose 50 values in mice were 5,000 and 2,609 mg/kg for essential oil and citronellal, respectively. E. citriodora essential oil reduced sheep epg at 14 days post treatment by 69.5% (P<0.05). No significant differences were observed in epg between the citronellal and negative control groups (P>0.05). The interaction between citronellal and other constituents in the essential oil may be relevant for its in vivo anthelmintic activity. Thus, E. citriodora essential oil and citronellal pharmacokinetic studies may help elucidate the anthelmintic activity of these compounds.

Anthelmintic activity of Eucalyptus citriodora essential oil and its major component, citronellal, on sheep gastrointestinal nematodes / Atividade anti-helmíntica do óleo essencial de Eucalyptus citriodora e seu componente majoritário, citronelal, sobre nematoides gastrintestinais de ovinos

Abstract This study aimed to evaluate the anthelmintic activity of Eucalyptus citriodora essential oil and citronellal on sheep gastrointestinal nematodes. Essential oil composition was determined by gas chromatography mass spectrometry. The substances were evaluated in vitro using adult worm motility test (AWMT) and transmission electron microscopy (TEM). The acute toxicity test in mice and the fecal egg count reduction test (FECRT) in sheep were performed. Citronellal was confirmed as the essential oil major constituent (63.9%). According to the AWMT, 2 mg/mL of essential oil and citronellal completely inhibited Haemonchus contortus motility at 6 h post exposure. H. contortus exposed to essential oil and citronellal exhibited internal ultrastructural modifications. The lethal dose 50 values in mice were 5,000 and 2,609 mg/kg for essential oil and citronellal, respectively. E. citriodora essential oil reduced sheep epg at 14 days post treatment by 69.5% (P<0.05). No significant differences were observed in epg between the citronellal and negative control groups (P>0.05). The interaction between citronellal and other constituents in the essential oil may be relevant for its in vivo anthelmintic activity. Thus, E. citriodora essential oil and citronellal pharmacokinetic studies may help elucidate the anthelmintic activity of these compounds.

Resumo Este trabalho objetivou avaliar a atividade anti-helmíntica do óleo essencial de Eucalyptus citriodora e citronelal sobre nematoides gastrintestinais de ovinos. A composição do óleo essencial foi determinada por cromatografia gasosa acoplada à espectrometria de massas. As substâncias foram avaliadas in vitro utilizando-se teste de motilidade de vermes adultos (AWMT) e microscopia eletrônica de transmissão (TEM). Teste de toxicidade aguda em camundongos e teste de redução da contagem de ovos fecais (FECRT) em ovinos foram realizados. Citronelal foi confirmado como componente majoritário do óleo essencial (63,9%). No AWMT, 2 mg/mL de óleo essencial e citronelal inibiram completamente a motilidade de H. contortus 6 h pós-exposição. H. contortus expostos ao óleo essencial e citronelal exibiram modificações ultraestruturais internas. Os valores da dose letal 50 em camundongos foram 5.000 e 2.609 mg/kg para óleo essencial e citronelal, respectivamente. Óleo essencial de E. citriodora reduziu opg de ovinos 14 dias pós-tratamento em 69,5% (P<0,05). Não houve diferença significativa de opg entre grupo controle negativo e citronelal (P>0,05). A interação entre citronelal e outros constituintes do óleo essencial pode ser relevante na atividade anti-helmíntica in vivo. Portanto, avaliação farmacocinética do óleo essencial de E. citriodora e citronelal pode auxiliar a elucidar a atividade anti-helmíntica desses compostos.

Anthelmintic effect of Cymbopogon citratus essential oil and its nanoemulsion on sheep gastrointestinal nematodes.

The anthelmintic resistance stimulated the search for strategies for controlling gastrointestinal nematodes, including the use of free essential oils or its nanoemulsion. This study evaluated the anthelmintic efficacy of Cymbopogon citratus essential oil (CcEO) and C. citratus essential oil nanoemulsion (CcEOn). Pysicochemical analyses were performed. The in vitro effect was determined using the egg hatch test (EHT) on Haemonchus contortus and in vivo effect was evaluated in sheep infected with gastrointestinal nematodes. The animals were treated with CcEO (500 mg/kg) or CcEOn (450 mg/kg) for the fecal egg count (FEC) and the determination of worm burden. The main component of CcEO was citral. The CcEO content in the nanoemulsion was 20% (v/v), and the mean particle size was 248 nm. In EHT, CcEO and CcEOn (1.25 mg/mL) inhibited larval hatching by 98.4 and 97.1%, respectively. Three animals treated with CcEO died whereas in the group treated with CcEOn one animal died. The FEC and total worm burden of the treated groups did not differ from the negative control (p>0.05). The CcEOn showed efficacy only on H. contortus (p<0.05). In conclusion, nanoencapsulation reduced toxicity and increased efficacy on H. contortus.

Anthelmintic effect of Cymbopogon citratus essential oil and its nanoemulsion on sheep gastrointestinal nematodes / Efeito anti-helmíntico do óleo essencial de Cymbopogon citratus e sua nanoemulsão sobre nematoides gastrintestinais de ovinos

Abstract The anthelmintic resistance stimulated the search for strategies for controlling gastrointestinal nematodes, including the use of free essential oils or its nanoemulsion. This study evaluated the anthelmintic efficacy of Cymbopogon citratus essential oil (CcEO) and C. citratus essential oil nanoemulsion (CcEOn). Pysicochemical analyses were performed. The in vitro effect was determined using the egg hatch test (EHT) on Haemonchus contortus and in vivo effect was evaluated in sheep infected with gastrointestinal nematodes. The animals were treated with CcEO (500 mg/kg) or CcEOn (450 mg/kg) for the fecal egg count (FEC) and the determination of worm burden. The main component of CcEO was citral. The CcEO content in the nanoemulsion was 20% (v/v), and the mean particle size was 248 nm. In EHT, CcEO and CcEOn (1.25 mg/mL) inhibited larval hatching by 98.4 and 97.1%, respectively. Three animals treated with CcEO died whereas in the group treated with CcEOn one animal died. The FEC and total worm burden of the treated groups did not differ from the negative control (p>0.05). The CcEOn showed efficacy only on H. contortus (p<0.05). In conclusion, nanoencapsulation reduced toxicity and increased efficacy on H. contortus.

Resumo A resistência anti-helmíntica estimulou a busca por estratégias de controle de nematoides gastrintestinais, incluindo óleos essenciais livres ou em nanoemulsão. Este estudo avaliou a eficácia anti-helmíntica do óleo essencial de Cymbopogon citratus (OECc) e da nanoemulsão do óleo essencial de C. citratus (nOECc). Análises físico-químicas foram realizadas. O efeito in vitro foi determinado no teste de eclosão de ovos (TEO) sobre Haemonchus contortus e o efeito in vivo foi avaliado em ovinos infectados com nematoides gastrintestinais. Os animais foram tratados com OECc (500 mg/kg) ou nOECc (450 mg/kg) para determinação do número de ovos por grama de fezes (OPG) e carga parasitária. O principal constituinte do OECc foi citral. O conteúdo de OECc na nanoemulsão foi 20% e o tamanho médio de partícula foi 248 nm. No TEO, OECc e nOECc (1,25 mg/mL) inibiram 98,4 e 97,1% da eclosão larvar, respectivamente. Três animais tratados com o OECc morreram, enquanto um animal do grupo tratado com a nOECc morreu. O OPG e a carga parasitária total dos grupos tratados não diferiram do controle negativo (p>0.05). A nOECc apresentou eficácia somente sobre H. contortus (p<0.05). Como conclusão, o nanoencapsulamento reduziu a toxicidade e aumentou a eficácia sobre H. contortus.

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

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 LD50 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.

Anthelmintic effect of thymol and thymol acetate on sheep gastrointestinal nematodes and their toxicity in mice / Efeito anti-helmíntico do timol e acetato de timila sobre nematoides gastrintestinais de ovinos e toxicidade em camundongos

Abstract 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 LD50 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.

Resumo Timol 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.

Anthelmintic activity of Cymbopogon citratus against Haemonchus contortus.

Parasitic nematodes are of major economic importance in livestock. An alternative for the control of parasites is phytotherapy. This study evaluated the efficacy of Cymbopogon citratus decoction (CcD), C. citratus essential oil (CcEo) and citral against Haemonchus contortus using in vitro egg hatch test (EHT) and larval development test (LDT) and an in vivo test using a Meriones unguiculatus (gerbil) model. The effect of 800 mg/kg CcEo was evaluated in gerbils that had been artificially infected with 5,000 third-stage H. contortus larvae. The effective concentrations required to inhibit 50% (EC50) of egg hatching were 0.46, 0.14 and 0.13 mg/mL for CcD, CcEo and citral, respectively. The EC50 values in the LDT were 5.04, 1.92 and 1.37 mg/mL for CcD, CcEo and citral, respectively. H. contortus population in the group treated with C. citratus essential oil was reduced by 38.5% (P< 0.05) in comparison to the control group. These results suggest that it may be possible to use C. citratus essential oil to control of H. contortus parasite of small ruminant.

Anthelmintic activity of Cymbopogon citratus against Haemonchus contortus / Atividade anti-helmíntica de Cymbopogon citratussobre Haemonchus contortus

Parasitic nematodes are of major economic importance in livestock. An alternative for the control of parasites is phytotherapy. This study evaluated the efficacy of Cymbopogon citratus decoction (CcD), C. citratus essential oil (CcEo) and citral against Haemonchus contortus using in vitro egg hatch test (EHT) and larval development test (LDT) and an in vivo test using a Meriones unguiculatus (gerbil) model. The effect of 800 mg/kg CcEo was evaluated in gerbils that had been artificially infected with 5,000 third-stage H. contortus larvae. The effective concentrations required to inhibit 50% (EC50) of egg hatching were 0.46, 0.14 and 0.13 mg/mL for CcD, CcEo and citral, respectively. The EC50 values in the LDT were 5.04, 1.92 and 1.37 mg/mL for CcD, CcEo and citral, respectively. H. contortus population in the group treated with C. citratus essential oil was reduced by 38.5% (P< 0.05) in comparison to the control group. These results suggest that it may be possible to use C. citratusessential oil to control of H. contortus parasite of small ruminant.

O parasitismo por nematoides tem grande importância econômica no rebanho. Uma alternativa para o controle de parasitas é a fitoterapia. Este estudo avaliou a eficácia do decocto de Cymbopogon citratus (DCc), do óleo essencial de C. citratus (OECc) e do citral contra Haemonchus contortus utilizando o teste in vitro de eclosão dos ovos (TEO) e o teste de desenvolvimento larval (TDL) e um teste in vivo com modelo Meriones unguiculatus (gerbil). O efeito de 800 mg/kg de OECc foi avaliado em gerbils infectados artificialmente com 5000 larvas de terceira fase de H. contortus. As concentrações efetivas necessárias para inibir 50% (CE50) da eclosão dos ovos foram 0,46; 0,14 e 0,13 mg/mL para DCc, OECc e citral, respectivamente. Os valores da CE50 no TDL foram de 5,04; 1,92 e 1,37 mg/mL para DCc, OECc e citral, respectivamente. No grupo tratado com óleo de C. citratus a população H. contortus foi reduzida em 38,5% (P < 0,05) em comparação com o grupo controle. Estes resultados sugerem que pode ser possível a utilização de óleo essencial de C. citratus para controle de H. contortus, parasita de pequenos ruminantes.

In vitro effects of Eucalyptus staigeriana nanoemulsion on Haemonchus contortus and toxicity in rodents.

Strategies for controlling gastrointestinal nematodes have been developed based on the use of numerous alternative methods, including the use of phytotherapy. New formulations of essential oils with anthelmintic activity have been proposed as a means to optimize their biological effects. Thus, the objective of this study was to formulate a nanoemulsion to optimize the nematicide effect of Eucalyptus staigeriana essential oil (EsEO). Initially, physico-chemical analyses were performed to verify the stability of the E. staigeriana nanoemulsion (EsNano). In vitro tests were conducted to evaluate the ovicidal and larvicidal activities of both EsNano and EsEO against Haemonchus contortus, and toxicology tests were then performed on rodents. The EsEO content in the nanoemulsion was 36.4% (v/v), and the mean particle size was 274.3 nm. EsNano and EsEO inhibited larval hatching by 99% and 96.3% at 1 and 2mg/ml concentrations, respectively, and inhibited larval development by 96.3% and 97.3% at 8 mg/ml concentrations. The acute toxicity test revealed that the EsNano and EsEO doses required to kill 50% of the mice (LD50) were 1,603.9 and 3,495.9 mg/ml, respectively. EsNano did not alter the hematological parameters in the rats after treatment.

Identification and quantification of benzimidazole resistance polymorphisms in Haemonchus contortus isolated in Northeastern Brazil.

Haemonchus contortus is the most prevalent nematode in Brazil. The objective of this study was to select 6 populations of H. contortus of known or suspected benzimidazole resistance status and characterize these using quantitative real-time polymerase chain reaction (qPCR) for single nucleotide polymorphisms (SNPs) F200Y, F167Y and E198A in the ß-tubulin isotype 1 gene. qPCR was performed using DNA from a pool of 10 adult male H. contortus from a single animal per farm. Faecal egg count reduction test (FECRT) and egg hatch test (EHT) were used to determine the resistance status. Samples were obtained from 6 farms located in 5 counties in the Ceará State: Tauá, Boa Viagem, Quixadá, Santa Quitéria and Solonópole. The inbred-susceptible-Edinburgh (ISE) isolate was used as reference for comparative purposes in the qPCR. Benzimidazole resistance was detected by FECRT on all farms with efficacy values ranging from 0 to 51%. EC50 values as determined by EHT were all above 1.49µg/ml. High frequencies of the resistant SNPs F200Y and F167Y alleles were detected but no resistance was detected at SNP E198A. Our results suggest that the SNPs F167Y and F200Y are both important for benzimidazole resistance in the studied populations.

In vitro effects of Coriandrum sativum, Tagetes minuta, Alpinia zerumbet and Lantana camara essential oils on Haemonchus contortus / Efeitos in vitro dos óleos essenciais de Coriandrum sativum, Tagetes minuta, Alpinia zerumbet e Lantana camara sobre Haemonchus contortus

Phytotherapy can be an alternative for the control of gastrointestinal parasites of small ruminants. This study evaluated the efficacy of Alpinia zerumbet, Coriandrum sativum, Tagetes minuta and Lantana camara essential oils by two in vitro assays on Haemonchus contortus, an egg hatch test (EHT) and larval development test (LDT). No effect was observed for L. camara in the EHT. A. zerumbet, C. sativum and T. minuta essential oils exhibited a dose-dependent effect in the EHT, inhibiting 81.2, 99 and 98.1% of H. contortus larvae hatching, respectively, at a concentration of 2.5 mg mL-¹. The effective concentration to inhibit 50% (EC50) of egg hatching was 0.94, 0.63 and 0.53 mg mL-¹ for A. zerumbet, C. sativum and T. minuta essential oils, respectively. In LDT, L. camara, A. zerumbet, C. sativum and T. minuta at concentration of 10 mg mL-¹ inhibited 54.9, 94.2, 97.8 and 99.5% of H. contortus larval development, presenting EC50 values of 6.32, 3.88, 2.89 and 1.67 mg mL-¹, respectively. Based on the promising results presented in this in vitro model, it may be possible use of these essential oils to control gastrointestinal nematodes. However, their anthelmintic activity should be confirmed in vivo.

Fitoterapia pode ser uma alternativa para o controle de parasitas gastrintestinais de pequenos ruminantes. Este estudo avaliou a eficácia dos óleos essenciais de Alpinia zerumbet, Coriandrum sativum, Tagetes minuta e Lantana camara sobre Haemonchus contortus através de dois testes in vitro, teste de eclosão dos ovos (TEO) e teste de desenvolvimento larval (TDL). Nenhum efeito foi observado para L. camara no TEO. Os óleos essenciais de A. zerumbet, C. sativum e T. minuta exibiram um efeito dose dependente no TEO inibindo a eclosão das larvas de H. contortus em 81,2, 99 e 98,1%, respectivamente, na concentração de 2,5 mg mL-¹. A concentração efetiva para inibir 50% (CE50) da eclosão dos ovos foi de 0,94, 0,63 e 0,53 mg mL-¹ para os óleos essenciais de A. zerumbet, C. sativum e T. minuta, respectivamente. No TDL, 10 mg mL-¹ de L. camara, A. zerumbet, C. sativum e T. minuta inibiram em 54,9, 94,2, 97,8 e 99,5% do desenvolvimento larvar, apresentando valores de CE50 de 6,32, 3,88, 2,89 e 1,67 mg mL-¹, respectivamente. Com base nos resultados promissores apresentados neste modelo in vitro, pode ser possível a utilização destes óleos essenciais para controlar os nematoides gastrintestinais. No entanto, a sua atividade anti-helmíntica deve ser confirmada in vivo.

Activity of chitosan-encapsulated Eucalyptus staigeriana essential oil on Haemonchus contortus.

Phytotherapy has been described as an alternative method for the control of gastrointestinal nematodes in small ruminants. Goal of the encapsulation of essential oils in biopolymer matrices is to optimize the biological effects of these oils. The aim of the present study was to evaluate the in vitro and in vivo anthelmintic activity of encapsulated Eucalyptus staigeriana essential oil (EncEs) on the eggs and larvae of Haemonchus contortus. Therefore, the egg hatching test (EHT), larval development test (LDT) and worm load evaluation were performed to evaluate Meriones unguiculatus experimentally infected with H. contortus. The chemical constituents of E. staigeriana essential oil (EsEO) and the in vitro oil release profile from the chitosan matrix at a pH of 1.2 and 7.0 were also characterized. EncEs and EsEO inhibited larval hatching by 97.19% and 99.96% at doses of 1.5 and 1.0 mg ml(-1), respectively. In the LDT, EncEs and EsEO induced a larvicidal effect greater than 95% at concentrations of 5.8 and 8 mg ml(-1), respectively. EncEs and EsEO decreased H. contortus load in M. unguiculatus by 40.51% and 46.44%, respectively. The major chemical constituents of EsEO were (+)-Limonene (72.9%), 1,8-Cineole (9.5%) and o-Cimene (4.6%). The release profile of EsEO was 30% in acid and 25% at neutral pH. The similar efficacy of EncEs and EsEO demonstrates that there was no optimization of anthelmintic action following the encapsulation process. Therefore, the use of new encapsulation matrices with controlled release in the pH of the abomasum should be investigated.

In vitro effects of Coriandrum sativum, Tagetes minuta, Alpinia zerumbet and Lantana camara essential oils on Haemonchus contortus.

Phytotherapy can be an alternative for the control of gastrointestinal parasites of small ruminants. This study evaluated the efficacy of Alpinia zerumbet, Coriandrum sativum, Tagetes minuta and Lantana camara essential oils by two in vitro assays on Haemonchus contortus, an egg hatch test (EHT) and larval development test (LDT). No effect was observed for L. camara in the EHT. A. zerumbet, C. sativum and T. minuta essential oils exhibited a dose-dependent effect in the EHT, inhibiting 81.2, 99 and 98.1% of H. contortus larvae hatching, respectively, at a concentration of 2.5 mg mL-1. The effective concentration to inhibit 50% (EC50) of egg hatching was 0.94, 0.63 and 0.53 mg mL-1 for A. zerumbet, C. sativum and T. minuta essential oils, respectively. In LDT, L. camara, A. zerumbet, C. sativum and T. minuta at concentration of 10 mg mL-1 inhibited 54.9, 94.2, 97.8 and 99.5% of H. contortus larval development, presenting EC50 values of 6.32, 3.88, 2.89 and 1.67 mg mL-1, respectively. Based on the promising results presented in this in vitro model, it may be possible use of these essential oils to control gastrointestinal nematodes. However, their anthelmintic activity should be confirmed in vivo.

Plantas taniníferas e o controle de nematóides gastrintestinais de pequenos ruminantes / Tanniferous plants and control of gastrointestinal nematodes of small ruminants

Os nematóides gastrintestinais são responsáveis por acentuada redução na produtividade de ovinos e caprinos. Na tentativa de controlar esses parasitos, são utilizados diversos anti-helmínticos sintéticos, mas o desenvolvimento da resistência tem tornado esse controle pouco eficaz. Na busca por alternativas de controle dos nematóides gastrintestinais, a utilização de plantas taniníferas tem despertado interesse de pesquisadores em várias regiões do mundo. Essa revisão visa a abordar os diversos aspectos relacionados ao fornecimento dessas plantas a ovinos e caprinos, bem como os principais resultados obtidos em pesquisas utilizando taninos para o controle de nematóides de pequenos ruminantes.

The gastrointestinal nematodes are responsible for a marked reduction in the productivity of sheep and goats. In an attempt to control these parasites, different synthetic anthelmintics are used, but development of resistance has become this control little effective. In the search for alternatives to control these nematodes, the use of tanniferous plants has attracted attention from researchers in different regions of the world. This review aims to address the various aspects related to the administration of these plants to sheep and goats, and the main results obtained from studies using tannins to control nematodes in small ruminants.

Evaluation of Eucalyptus citriodora essential oil on goat gastrointestinal nematodes.

Phytotherapy may be an alternative strategy for controlling gastrointestinal parasites. This study evaluated the anthelmintic efficacy of Eucalyptus citriodora essential oil (EcEO). The in vitro effects of EcEO were determined through testing the inhibition of egg hatching and larval development of Haemonchus contortus. EcEO was subjected to acute toxicity testing on mice, orally and intraperitoneally. The in vivo effects of EcEO were determined by the fecal egg count reduction test (FECRT) in goats infected with gastrointestinal nematodes. The results showed that 5.3 mg.mL(-1) EcEO inhibited egg hatching by 98.8% and 10.6 mg.mL(-1) EcEO inhibited H. contortus larval development by 99.71%. The lethal doses for 50% of the mice were 4153 and 622.8 mg.kg(-1), for acute toxicity orally and intraperitoneally. In the FECRT, the efficacy of EcEO and ivermectin was 66.25 and 79.16% respectively, on goat gastrointestinal nematodes eight days after treatment. EcEO showed in vitro and in vivo anthelmintic activity.

Evaluation of Eucalyptus citriodora essential oil on goat gastrointestinal nematodes / Avaliação do óleo essencial de Eucalyptus citriodora sobre nematóides gastrintestinais de caprinos

Phytotherapy may be an alternative strategy for controlling gastrointestinal parasites. This study evaluated the anthelmintic efficacy of Eucalyptus citriodora essential oil (EcEO). The in vitro effects of EcEO were determined through testing the inhibition of egg hatching and larval development of Haemonchus contortus. EcEO was subjected to acute toxicity testing on mice, orally and intraperitoneally. The in vivo effects of EcEO were determined by the fecal egg count reduction test (FECRT) in goats infected with gastrointestinal nematodes. The results showed that 5.3 mg.mL-1 EcEO inhibited egg hatching by 98.8 percent and 10.6 mg.mL-1 EcEO inhibited H. contortus larval development by 99.71 percent. The lethal doses for 50 percent of the mice were 4153 and 622.8 mg.kg-1, for acute toxicity orally and intraperitoneally. In the FECRT, the efficacy of EcEO and ivermectin was 66.25 and 79.16 percent respectively, on goat gastrointestinal nematodes eight days after treatment. EcEO showed in vitro and in vivo anthelmintic activity.

Fitoterapia pode ser uma estratégia alternativa para o controle de parasitas gastrintestinais. Este estudo avaliou a eficácia anti-helmintica do óleo essencial de Eucalyptus citriodora (OeEc). Os efeitos in vitro do OeEc foram determinados através do teste de eclosão de ovos e inibição do desenvolvimento larvar de Haemonchus contortus. O OeEc foi submetido ao teste de toxicidade aguda oral e intraperitoneal, em camundongos. Os efeitos in vivo do OeEc foram avaliados através do teste de redução da contagem de ovos nas fezes (FECRT) com caprinos infectados com nematóides gastrintestinais. Os resultados mostraram que 5,3 mg.mL-1 OeEc inibiram 98,8 por cento a eclosão de ovos e 10,6 mg.mL-1 OeEc inibiram 99,71 por cento o desenvolvimento larvar de H. contortus. As doses letais para 50 por cento dos camundongos foram de 4153 e 622,8 mg.kg-1 pela via oral e intraperitoneal. No FECRT, a eficácia de OeEc e ivermectina foi de 66,25 e 79,16 por cento, respectivamente, em caprinos 8 dias após o tratamento. OeEc mostrou atividade anti-helmintica in vitro e in vivo.

Effect of six tropical tanniferous plant extracts on larval exsheathment of Haemonchus contortus.

Tanniferous plants represent a promising alternative for controlling gastrointestinal nematodes of small ruminants. This experiment evaluated the effects of extracts from the leaf and stem of Anadenanthera colubrina, Leucaena leucocephala and Mimosa tenuiflora on larval exsheathment of Haemonchus contortus in vitro and verified the role of tannins in this process. Third-stage larvae of H. contortus were incubated with extracts for 3 hours and were exposed to sodium hypochlorite solution. The extracts were tested at 300 µg.mL(-1) and accompanied by controls: phosphate buffer solution (PBS) and polyvinyl polypyrrolidone (PVPP). The larval exsheathment was evaluated for 60 minutes, and the results were subjected to the Kruskal-Wallis test (p < 0.05). The six extracts blocked larval exsheathment. After PVPP addition, a tannin inhibitor, the exsheathment percentage was similar to the PBS (p > 0.05), except for L. leucocephala and M. tenuiflora leaf extracts. However, pre-incubation with PVPP of these two extracts significantly changed larval exsheathment when compared to the non-treated extracts (p < 0.05). These results suggest that A. colubrina, L. leucocephala and M. tenuiflora could be useful in gastrointestinal nematode control and that tannins are probably the main compounds involved in the observed effects. However, in vivo and toxicological studies should be conducted.

Effect of six tropical tanniferous plant extracts on larval exsheathment of Haemonchus contortus / Efeito de seis extratos de plantas taniníferas tropicais sobre o desembainhamento larvar de Haemochus contortus

Tanniferous plants represent a promising alternative for controlling gastrointestinal nematodes of small ruminants. This experiment evaluated the effects of extracts from the leaf and stem of Anadenanthera colubrina, Leucaena leucocephala and Mimosa tenuiflora on larval exsheathment of Haemonchus contortus in vitro and verified the role of tannins in this process. Third-stage larvae of H. contortus were incubated with extracts for 3 hours and were exposed to sodium hypochlorite solution. The extracts were tested at 300 µg.mL-1 and accompanied by controls: phosphate buffer solution (PBS) and polyvinyl polypyrrolidone (PVPP). The larval exsheathment was evaluated for 60 minutes, and the results were subjected to the Kruskal-Wallis test (p < 0.05). The six extracts blocked larval exsheathment. After PVPP addition, a tannin inhibitor, the exsheathment percentage was similar to the PBS (p > 0.05), except for L. leucocephala and M. tenuiflora leaf extracts. However, pre-incubation with PVPP of these two extracts significantly changed larval exsheathment when compared to the non-treated extracts (p < 0.05). These results suggest that A. colubrina, L. leucocephala and M. tenuiflora could be useful in gastrointestinal nematode control and that tannins are probably the main compounds involved in the observed effects. However, in vivo and toxicological studies should be conducted.

Plantas taniníferas representam uma promissora alternativa de controle dos nematóides gastrintestinais de pequenos ruminantes. Esse experimento avaliou in vitro os efeitos dos extratos das folhas e caules de Anadenanthera colubrina, Leucaena leucocephala e Mimosa tenuiflora sobre o desembainhamento larvar de Haemonchus contortus e verificou o papel dos taninos nesse processo. Larvas de terceiro estádio de H. contortus foram incubadas com 300 µg.mL-1 de extrato por 3 horas e expostas a uma solução de hipoclorito de sódio. O ensaio foi acompanhado por controles: solução salina tamponada com fosfato (PBS) e polivinilpolipirrolidona (PVPP). O desembainhamento larvar foi avaliado durante 60 minutos e os resultados submetidos ao teste Kruskal-Wallis (p < 0,05). Os seis extratos bloquearam o desembainhamento larvar. Após adição de PVPP, um inibidor de taninos, o percentual de desembainhamento foi similar ao PBS (p > 0,05), exceto nos extratos das folhas de L. leucocephala e M. tenuiflora. Entretanto, a pré-incubação desses dois extratos com PVPP alterou significativamente o desembainhamento quando comparado com extratos não-tratados (p < 0,05). Esses resultados sugerem que A. colubrina, L. leucocephala e M. tenuiflora podem ser úteis no controle de nematóides gastrintestinais e que os taninos são provavelmente os principais compostos envolvidos nos efeitos. Contudo, estudos toxicológicos e in vivo são necessários.

Effects of Myracrodruon urundeuva extracts on egg hatching and larval exsheathment of Haemonchus contortus.

The anthelmintic resistance has limited the control of gastrointestinal nematodes of small ruminants and thus has awakened interest in the study of tanniferous plants as a source of anthelmintics. These experiments were carried out to evaluate the in vitro efficacy of Myracrodruon urundeuva leaf extract (LE) and stem extract (SE) against Haemonchus contortus. An inhibitor of tannins, polyvinyl polypyrrolidone (PVPP), was used to verify if these metabolites are involved in the anthelmintic activity of the extracts. To evaluate the ovicidal effect, H. contortus eggs were incubated with the extracts (0.31 to 5 mg/mL) for 48 h. In the larval artificial exsheathment assay, third-stage larvae of this nematode were incubated with extracts (0.31 mg/mL) for 3 h and then were exposed to a sodium hypochlorite solution. The exsheathment process was evaluated for 60 min. The results were subjected to the Kruskal-Wallis test (P < 0.05). The extracts showed dose-dependent ovicidal effects, although the LE was more effective, inhibiting egg hatching by 97.73% at 1.25 mg/mL, while the SE inhibited hatching by 83.56% at 5 mg/mL. Contact with the extracts blocked the larval exsheathment (P < 0.05). The addition of PVPP confirmed the role of tannins, as there was a substantial reduction in egg hatching and larval exsheathment percentage. These results suggest that M. urundeuva can be used to control gastrointestinal nematodes of small ruminants and that the anthelmintic activity of this plant is probably related to tannins; however, in vivo studies should be conducted.