Chemical characterization and in vitro biological activity of Cymbopogon citratus extracts against Haemonchus spp. and Trichostrongylus spp. nematodes from sheep.

Medicinal plants have been the focus of several studies due to their nematicide properties which can be used to control nematodes in sheep. No study has examined the morphological effects of Cymbopogon citratus on nematodes. Thus, this study evaluated the chemical composition, nematicidal activity and effects of C. citratus extracts on the morphology of eggs and infective larvae (L3) of sheep. Aqueous and methanolic extracts and fractions of C. citratus were obtained and analysed in vitro. The C. citratus extracts were effective against Haemonchus spp. and Trichostrongylus spp. larvae and eggs. Ten fractions were obtained from C. citratus, six of which had high ovicidal activity at 1000 µg mL-1, and two fractions had high activity at all tested concentrations. The phytochemical analysis identified the presence of compounds such as terpenoids, various ketones, esters, and fatty acids. The ultrastructural analysis showed deformations of the cuticle and wilting along the body of the nematodes at all concentrations. The muscular layer, intestinal cells and the mitochondria profile showed damage compared to the typical pattern. Ultra-thin sections of eggs treated with methanolic fractions of C. citratus presented modifications. This study showed the biological activity and effects of C. citratus on the gastrointestinal nematodes in sheep.

Economic viability of anthelmintic treatment in naturally infected beef cattle under different nutritional strategies after weaning.

The aim of the present study was to evaluate the effects of treatment with different anthelmintic compounds on the productivity of naturally infected calves and the economic viability of these treatments within extensive breeding systems employing different nutritional strategies after weaning. For this purpose, 4 farms with 42-60 calves naturally infected with gastrointestinal nematodes were selected. The calves were distributed into 6 groups (7-10 animals each) per farm and treated with ivermectin 1%, ivermectin 3.15%, eprinomectin 5%, levamisole 7.5%, albendazole 15%, and control group (no treatment). These animals were evaluated over an experimental period of 150 days. Levamisole 7.5% presented the best capacity for the reduction of eggs per gram (EPG) of feces in all herds evaluated, followed by albendazole 15% and eprinomectin 5%. Parasite resistance to multiple drugs was found in all herds, especially those of Cooperia, Haemonchus, Oesophagostomum, and Trichostrongylus. For farm 1, differences in weight gain and EPG reduction percentages led to a difference of US$285.06 between the levamisole and ivermectin 3.15% groups. Similar findings were noted for the levamisole and ivermectin 1% groups of farm 3, with a difference of US$399.37 because of the final weight gain in these groups. For farms 2 and 4, the ivermectin 3.15% and control groups, respectively, were the most profitable; these unexpected results were possibly influenced by variables not measured during the experimental period. This study suggested that anthelmintic treatments should always precede an efficacy test, once they are demonstrated to be most profitable under adequate breeding conditions, to ensure adequate control of gastrointestinal nematode infection.

Monepantel resistant Trichostrongylus colubriformis in goats in Brazil.

In a farm where Trichostrongylus colubriformis in sheep had been found resistant to monepantel (MNP), we aimed to evaluate the efficacy against gastrointestinal nematodes in a controlled efficacy test (CT) and a fecal egg count reduction test (FECRT) in goats. We used 15 (CT) and 30 goats (FECRT), naturally infected, and equally divided into control and treated groups. The efficacy of MNP in the CT against Haemonchus contortus, Trichostrongylus axei and Cooperia curticei was 100%. For T. colubriformis, the efficacy was 90.5% in goats treated with 2.5mg/kg, and 88.1% in goats treated with 3.5mg/kg. In the FECRT, the overall reduction was 91% (2.5mg/kg) and 90% (3.75mg/kg). In the fecal cultures, Trichostrongylus spp. larvae represented 25% (pre-treatment) and 15% (post-treatment) of the larvae in the control group, but they were 75% (2.5mg/kg) and 98% (3.75mg/kg) of the recovered larvae after MNP treatment. In accordance with published standards, and based on FECRT and CT, MNP was considered ineffective against T. colubriformis in the studied flock.

Ovicidal and larvicidal activity of extracts of Opuntia ficus-indica against gastrointestinal nematodes of naturally infected sheep.

This study describes the in vitro anthelmintic activity of extracts from Opuntia ficus indica against gastrointestinal nematodes of sheep. The anthelmintic activity was evaluated by inhibition of egg hatching, larval development and larval migration assays. The residual aqueous fractions from cladodes and fruits showed higher ovicidal activity with EC50 values of 7.2mg/mL and 1.5mg/mL, respectively. The aqueous, hexane, and ethyl acetate fractions from fruits and the aqueous fraction from cladodes inhibited 100% of larval development at the lowest concentration tested (1.56mg/mL). The crude cladode and fruit ethanolic extracts inhibited larval migration and showed EC50 values of 0.74mg/mL and 0.27mg/mL, respectively. Phytochemical screening detected high concentrations of alkaloids, tannins, flavonoids, and saponins in the fruits and cladodes. The results demonstrated that O. ficus exhibits anthelmintic activity in vitro, suggesting that, beyond its nutritional potential, this plant can also be an ally for parasite control in sheep.

Lack of efficacy of monepantel against Trichostrongylus colubriformis in sheep in Brazil.

Multiple drug resistance of nematodes against anthelmintics has become one of the most important economic problems in sheep production worldwide. The aim of this experiment was to evaluate the efficacy of monepantel (2.5mg/kg) against gastrointestinal nematodes in fecal egg count reduction test (FECRT) and controlled efficacy test (CT) in naturally infected sheep. We used 30 sheep for the FECRT and 20 sheep for the CT, equally divided into control and treated groups. In the FECRT, the reduction was 98%. Larval identification of pre-treatment coprocultures revealed 100% Haemonchus spp. for both control and treated groups. Post-treatment culture of treated sheep was 100% Oesophagostomum spp., but only few larvae were recovered. In the control group, they were 99% Haemonchus spp and 1% Oesophagostomum spp. larvae. Based on the FECRT, Haemonchus spp. was considered susceptible to monepantel. The efficacy of monepantel in the CT against Haemonchus contortus and Trichostrongylus axei was 100% and against Cooperia curticei was 99.7%. For Trichostrongylus colubriformis, the efficacy was -21.5%. In both treated and untreated animals, Oesophagostomum columbianum was recovered from the large intestines. Based on FECRT and CT and in accordance with WAAVP standards, monepantel was ineffective against T. colubriformis and O. columbianum, but effective against H. contortus, T. axei and C. curticei in the studied flock.

Dried, ground banana plant leaves (Musa spp.) for the control of Haemonchus contortus and Trichostrongylus colubriformis infections in sheep.

To evaluate the anthelmintic effect of Musa spp. leaves, 12 animals were artificially infected with Haemonchus contortus, and another 12 animals were infected with Trichostrongylus colubriformis. Then, both treatment groups were offered 400 g of dried ground banana plant leaves, and the control animals were offered only 1000 g of coast cross hay. During the trials, the animals received weekly physical examinations. The methods used to evaluate the efficiency of this treatment were packed cell volume, total plasma protein and faecal egg counts, and egg hatchability tests were performed on days -2, +3, +6, +9, +13 and +15. Coproculture tests were performed on day -2 to confirm monospecific infections. In the FEC and EHT, a statistically significant difference (0.04, 0.005; p < 0.05) was noted for T. colubriformis. There were no statistically significant differences (p > 0.05) for Haemochus contortus group in all tests. Our results confirmed previous findings suggesting that dried ground banana plant leaves possess anthelmintic activity.

Resistance of Haemonchus, Cooperia, Trichostrongylus, and Oesophagostomum to ivermectin in dairy cattle in Paraná / Resistência de Haemonchus, Cooperia, Trichostrongylus e Oesophagostomum à ivermectina em bovinos de leite no Paraná

The objective of this study was to analyze the effect of ivermectin (3.15%) on gastrointestinal nematodes found in calves through the fecal egg count reduction test (FECRT), and larval culture in 16 animals from a school farm at the Universidade Estadual do Norte do Paraná. The animals were divided into two experimental groups: animals treated with ivermectin 3.15% (G1 group) and control animals (G2 group). Stool samples were collected from the animals twice i.e., on days 0 and 10 for fecal examinations. There was no significant change in the egg count per gram of feces (EPG) in both the groups. Helminths were found to be resistant to ivermectin (3.15%), due to the observed 5.62% FECRT rate. Cooperia was found to be the dominant nematode larvae present (53% on day 10 day and 8% in day 0), while Trichostrongylus was detected in small numbers (0% on day 0 to 1 % on day 10). The drug was found to be effective only in Trichuris, while the same was inefficient against Haemonchus and Cooperia, although Trichostrongylus and Oesophagostomum were found to be 100 % resistant. Therefore, the findings of this study suggest that ivermectin 3.15% should not be used for the control of nematodes in calves, since most of the parasites demonstrated drug resistance. This is the first report on the resistance of Haemonchus, Cooperia, Trichostrongylus, and Oesophagostomum in cattle to ivermectin 3.(AU)

Objetivou-se, neste estudo, analisar o efeito da ivermectina (3,15%) sobre nematódeos gastrointestinais de bezerros com aptidão leiteira por meio do Teste de Redução na Contagem de Ovos nas Fezes (RCOF) e da cultura de larvas em 16 animais da Fazenda Escola da Universidade Estadual do Norte do Paraná. Para tanto, dois grupos experimentais foram formados: animais tratados com ivermectina 3,15% (grupo G1) e grupo controle (Grupo G2). Foram coletadas amostras de fezes dos animais em dois momentos, ou seja, nos dias 0 e 10 para realização dos exames coproparasitológicos. Não houve alteração significativa na contagem de ovos nas fezes (OPG) em ambos os grupos. Os helmintos foram considerados resistentes à ivermectina 3,15%, já que a taxa de RCOF foi de 5,62%. O gênero de nematódeo mais observado na cultura de larvas foi Cooperia (53% no dia 10 a 88% no dia zero) e o menos foi Trichostrongylus (0% no dia zero a 1% no dia 10). Observou-se eficiência apenas sobre o gênero Trichuris e ineficiência sobre os gêneros Haemonchus e Cooperia e ainda 100% de resistência dos gêneros Oesophagostomum e Trichostrongylus. Concluiu-se neste estudo que a ivermectina 3,15% não deve ser indicada no controle de nematódeos dos bezerros da propriedade, pois a maioria dos parasitas apresenta resistência à droga. Este foi o primeiro relato de resistência de Haemonchus, Cooperia, Trichostrongylus e Oesophagostomumde bovinos a ivermectina 3,15% no estado do Paraná.(AU)

Resistance of Haemonchus, Cooperia, Trichostrongylus, and Oesophagostomum to ivermectin in dairy cattle in Paraná / Resistência de Haemonchus, Cooperia, Trichostrongylus e Oesophagostomum à ivermectina em bovinos de leite no Paraná

The objective of this study was to analyze the effect of ivermectin (3.15%) on gastrointestinal nematodes found in calves through the fecal egg count reduction test (FECRT), and larval culture in 16 animals from a school farm at the Universidade Estadual do Norte do Paraná. The animals were divided into two experimental groups: animals treated with ivermectin 3.15% (G1 group) and control animals (G2 group). Stool samples were collected from the animals twice i.e., on days 0 and 10 for fecal examinations. There was no significant change in the egg count per gram of feces (EPG) in both the groups. Helminths were found to be resistant to ivermectin (3.15%), due to the observed 5.62% FECRT rate. Cooperia was found to be the dominant nematode larvae present (53% on day 10 day and 8% in day 0), while Trichostrongylus was detected in small numbers (0% on day 0 to 1 % on day 10). The drug was found to be effective only in Trichuris, while the same was inefficient against Haemonchus and Cooperia, although Trichostrongylus and Oesophagostomum were found to be 100 % resistant. Therefore, the findings of this study suggest that ivermectin 3.15% should not be used for the control of nematodes in calves, since most of the parasites demonstrated drug resistance. This is the first report on the resistance of Haemonchus, Cooperia, Trichostrongylus, and Oesophagostomum in cattle to ivermectin 3.

Objetivou-se, neste estudo, analisar o efeito da ivermectina (3,15%) sobre nematódeos gastrointestinais de bezerros com aptidão leiteira por meio do Teste de Redução na Contagem de Ovos nas Fezes (RCOF) e da cultura de larvas em 16 animais da Fazenda Escola da Universidade Estadual do Norte do Paraná. Para tanto, dois grupos experimentais foram formados: animais tratados com ivermectina 3,15% (grupo G1) e grupo controle (Grupo G2). Foram coletadas amostras de fezes dos animais em dois momentos, ou seja, nos dias 0 e 10 para realização dos exames coproparasitológicos. Não houve alteração significativa na contagem de ovos nas fezes (OPG) em ambos os grupos. Os helmintos foram considerados resistentes à ivermectina 3,15%, já que a taxa de RCOF foi de 5,62%. O gênero de nematódeo mais observado na cultura de larvas foi Cooperia (53% no dia 10 a 88% no dia zero) e o menos foi Trichostrongylus (0% no dia zero a 1% no dia 10). Observou-se eficiência apenas sobre o gênero Trichuris e ineficiência sobre os gêneros Haemonchus e Cooperia e ainda 100% de resistência dos gêneros Oesophagostomum e Trichostrongylus. Concluiu-se neste estudo que a ivermectina 3,15% não deve ser indicada no controle de nematódeos dos bezerros da propriedade, pois a maioria dos parasitas apresenta resistência à droga. Este foi o primeiro relato de resistência de Haemonchus, Cooperia, Trichostrongylus e Oesophagostomumde bovinos a ivermectina 3,15% no estado do Paraná.

Fifteen years later, anthelmintic resistances have dramatically spread over goat farms in Guadeloupe.

Faecal egg count reduction tests (FECRTs) were performed on 21 goat farms in Guadeloupe (FWI). Anthelmintic resistance (AR) to netobimin (benzimidazole) was found in all 15 herds in which it was tested. AR to ivermectin (avermectin) and levamisole (imidazothiazole) were also very largely spread (14 out of 17 farms and 7 out of 9 farms, respectively). AR to the final moxidectin (milbemycin) released was already present in 2 out of 9 farms in which it was tested. Haemonchus was the dominant genus of gastrointestinal nematodes and was more frequently found to be resistant to netobimin, ivermectin and moxidectin than Trichostrongylus, the latter appeared to be more often resistant to levamisole. A first survey 15 years ago revealed only AR to benzimidazoles and one suspected case of AR to ivermectin.

Anthelmintic efficacy of five tropical native Australian plants against Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected goats (Capra hircus).

The study of the anthelmintic properties of plants rich in plant secondary metabolites can provide ecologically sound methods for the treatment of parasites on grazing animals. The purpose of the present study was to evaluate the anthelmintic effect of five tropical native Australian plant species rich in plant secondary metabolites on adult Haemonchus contortus and Trichostrongylus colubriformis in experimentally infected goats. Thirty young, nematode-free goats were infected with 2500 H. contortus and 5000 T. colubriformis infective larvae thrice weekly for a week (day 1-7 of the experiment). On day 27 after first infection, the goats were allocated into six groups of five animals per group. From day 28 to day 35, fresh leaves from Acacia salicina, Acacia nilotica, Eucalyptus corymbia, Casuarina cunninghamiana and Eucalyptus drepanophylla were included in the goats diet. Five groups were offered leaves from one of these plant species and one group, the untreated control, received only the basal diet formulated with 20% Medicago sativa and 80% Avena sativa. Following plant material administration, the goats were monitored daily until day 40 and then slaughtered on day 41. Total faecal worm egg output, total production of larvae recovered from faecal cultures, total post-mortem worm burdens and the per capita fecundity of female worms were estimated. The toxicity of the plant species for the goats was measured by histopathological analyses of liver and kidney samples. Results showed that goats feeding on the plant material rich in plant secondary metabolites had significantly lower egg output compared to the control goats (P<0.05). A similar response was found for larval production in both H. contortus and T. colubriformis supporting that egg output was affected in both species. Although the total worm burdens were not affected by the plant material (P>0.05), the per capita fecundity was significantly reduced by E. corymbia, A. nilotica and A. salicina (P<0.05). No signs of toxicity were detected in the liver or kidney samples. It is concluded that goats can benefit from the short-term ingestion of plant secondary metabolites, which reduce the total faecal egg output and thus decrease the potential for re-infection from the pasture.

Anthelmintic activity of Cymbopogon martinii, Cymbopogon schoenanthus and Mentha piperita essential oils evaluated in four different in vitro tests.

Anthelmintic resistance is a worldwide concern in small ruminant industry and new plant-derived compounds are being studied for their potential use against gastrointestinal nematodes. Mentha piperita, Cymbopogon martinii and Cymbopogon schoenanthus essential oils were evaluated against developmental stages of trichostrongylids from sheep naturally infected (95% Haemonchus contortus and 5% Trichostrogylus spp.) through the egg hatch assay (EHA), larval development assay (LDA), larval feeding inhibition assay (LFIA), and the larval exsheathment assay (LEA). The major constituent of the essential oils, quantified by gas chromatography for M. piperita oil was menthol (42.5%), while for C. martinii and C. schoenanthus the main component was geraniol (81.4% and 62.5%, respectively). In all in vitro tests C. schoenanthus essential oil had the best activity against ovine trichostrongylids followed by C. martini, while M. piperita presented the least activity. Cymbopogon schoenanthus essential oil had LC(50) value of 0.045 mg/ml in EHA, 0.063 mg/ml in LDA, 0.009 mg/ml in LFIA, and 24.66 mg/ml in LEA. The anthelmintic activity of essential oils followed the same pattern in all in vitro tests, suggesting C. schoenanthus essential oil could be an interesting candidate for nematode control, although in vivo studies are necessary to validate the anthelmintic properties of this oil.

Multiple resistance to anthelmintics by Haemonchus contortus and Trichostrongylus colubriformis in sheep in Brazil.

The objective of this study was to determine the level of resistance of Haemonchus contortus and Trichostrongylus colubriformis in sheep to levamisole, albendazole, ivermectin, moxidectin, closantel and trichlorfon. The parasites were isolated from sheep naturally infected by gastrointestinal nematodes and were then kept in monospecifically-infected lambs for production of infective larvae (L3) of both species. Forty-two lambs, at three months of age, were simultaneously artificially infected with 4000 L3 of H. contortus and 4000 L3 of T. colubriformis. The animals were allocated into seven groups with six animals each that received one of the following treatments: Group 1--control, no treatment; Group 2--moxidectin (0.2mg/kg body weight (BW)); Group 3--closantel (10mg/kg BW); Group 4--trichlorfon (100mg/kg BW); Group 5--levamisole phosphate (4.7 mg/kg BW); Group 6--albendazole (5.0mg/kg BW); and Group 7--ivermectin (0.2mg/kg BW). Nematode fecal egg counts (FEC) were carried out on the day of treatment and again at 3, 7, 10 and 14 days post-treatment. On the same occasions, composite fecal cultures were prepared for each group for production of L3, which were identified into genus. The animals were sacrificed for worm counts at 14 days after treatment. The efficacy of each treatment was calculated from the arithmetic mean of the FEC or worm burden of the treated group, compared with the values of the control group. Only trichlorfon and moxidectin treatments resulted in a significant reduction of H. contortus recorded at necropsy (73% and 45% respectively). Moxidectin reduced T. colubriformis worm burdens by 82% and albendazole by 19%. All other anthelmintics resulted in no significant reduction in the numbers of worms found at necropsy. In conclusion, the isolates of H. contortus and T. colubriformis showed multiple resistance to all groups of anthelmintics tested. This is the first report, based on the controlled efficacy test, to show resistance of T. colubriformis to macrocyclic lactones in Brazil.

Effects of four tropical tanniniferous plant extracts on the inhibition of larval migration and the exsheathment process of Trichostrongylus colubriformis infective stage.

The anthelmintic (AH) effect of Acacia pennatula, Leucaena leucocephala, Lisyloma latisiliquum and Piscidia piscipula was evaluated in the infective larvae (L(3)) of Trichostrongylus colubriformis. Different concentrations of lyophilized extracts were tested using the larval migration inhibition (LMI) test. An inhibitor of tannins (the polyvinyl polypyrrolidone [PVPP]) was used to verify whether these compounds were responsible for the AH effects. Then, the effect of extracts on larval exsheathment was examined by observing the exsheathment process at 10-min intervals for 70 min. The LMI test showed a dose-dependant AH effect for A. pennatula, L. leucocephala and L. latisiliquum (P<0.01), but not for P. piscipula. The restoration of L(3) migration to values similar to those of controls after the addition of PVPP, indicates that tannins are involved in AH effects. Trichostrongylus colubriformis exsheathment was partially or totally blocked by the four plants extracts. Tropical tanniniferous plants evaluated in the current study may have potential as AH for the control of T. colubriformis if in vivo investigations indicate useful effects.

Nematicidal alkylene resorcinols from Lithraea molleoides.

Four new alkylene resorcinols, (Z,Z)-5-(trideca-4,7-dienyl)resorcinol (1), (Z,Z,Z)-5-(trideca-4,7,10-trienyl)resorcinol (2), (Z,Z,E)-5-(trideca-4,7,10-trienyl)resorcinol (3), and (Z)-5-(trideca-4-enyl)resorcinol (4), were isolated from the MeOH-CH(2)Cl(2) extract of Lithraea molleoides. The structures of these compounds were determined by one- and two-dimensional NMR including selective decoupling experiments. In vitro all four compounds showed strong paralyzing effects on the nematode Caenorhabditis elegans at concentrations between 6 and 50 microg/mL, whereas the activity of compounds 1 and 2 against the nematode Trichostrongylus colubriformis was less pronounced and no activity against this nematode was observed for compounds 1-4 in a rodent model.

The prevalence of anthelmintic resistance in nematode parasites of sheep in southern Latin America: Paraguay.

This survey to detect anthelmintic resistance in nematode parasites of sheep was conducted on 11 farms in the Occidental and 26 farms in the Oriental Regions of Paraguay using the faecal egg count reduction test (FECRT). The anthelmintic groups tested were the benzimidazoles, levamisole and avermectins (both oral and injectable). Overall the levels of resistance were 73%, 68%, 73% and 47%, respectively. Levels of resistance were similar for all three important nematode genera, viz. Haemonchus contortus, Ostertagia and Trichostrongylus. This survey clearly indicates that a large, and ever increasing, proportion of sheep farmers are rapidly approaching the time when they will have exhausted all chemotherapeutic options to control parasites. Unless they face having to abandon their sheep farming operations, radical changes will need to be implemented with urgency.

The prevalence of anthelmintic resistance in nematode parasites of sheep in southern Latin America: Uruguay.

This survey was conducted on 252 farms randomly distributed over all the sheep raising areas in Uruguay. The study involved farms with more than 600 sheep, which represented 80% of the total sheep population of the country. Three anthelmintic groups were assessed, namely, benzimidazoles, levamisole and avermectins. Overall, the results showed 80% of sheep flocks had benzimidazole resistance, 71% had resistance to levamisole, and 1.2% of flocks showed resistance to avermectins. Approximately 28% of farms had resistance to one anthelmintic group, 64% to two anthelmintic groups, and 1% resistance to all three groups. Only 7.5% of farms had no detectable levels of anthelmintic resistance. More than 80% of farms had Trichostrongylus populations resistant to both benzimidazoles and levamisole. Resistance was recorded in all three anthelmintic groups for Haemonchus and resistance also occurred to benzimidazoles and levamisole in Ostertagia.

Efeito da administraçäo de oxfendazol, ivermectina e levamisol sobre os exames coproparasitológicos de ovinos / Survey of anthelmintic treatment with oxfendazole, ivermectin and levamisole in sheep flocks

O trabalho foi realizado em nove propriedades do estado de Säo Paulo, com o objetivo de verificar o efeito da administraçäo de oxfendazol, ivermectina e levamisol sobre os exames coproparasitológicos de ovinos. Em cada propriedade foram formados aleatóriamente quatro grupos de ovinos: o primeiro foi medicado com oxfendazol, na dose de 4,5 mg/kg, o segundo com levamisol, na dose de 7,5 mg/kg, o terceiro com ivermectina, na dose de 0,2 mg/kg e o quarto grupo foi o controle, näo medicado. Colheitas de fezes foram realizadas no dia da vermifugaçäo e novamente sete dias depois para a realizaçäo de exames coproparasitológicos. Após a administraçäo de oxfendazol, levamisol e ivermectina, foi verificada reduçäo estatística significativa nas contagens de ovos por grama de fezes (OPG) em duas, quatro e cinco propriedades, respectivamente, sendo Haemonchus e Trichostrongylus os parasitas com maior ocorrência no experimento. Os resultados sugerem, na maioria das propriedades, a presença de parasitas com resistência múltipla aos anti-helmínticos testados