Biomass-Based, Dual Enzyme-Responsive Nanopesticides: Eco-friendly and Efficient Control of Pine Wood Nematode Disease.

Pine wood nematode (PWN) disease is a globally devastating forest disease caused by infestation with PWN, Bursaphelenchus xylophilus, which mainly occurs through the vector insect Japanese pine sawyer (JPS), Monochamus alternatus. PWN disease is notoriously difficult to manage effectively and is known as the "cancer of pine trees." In this study, dual enzyme-responsive nanopesticides (AVM@EC@Pectin) were prepared using nanocoating avermectin (AVM) after modification with natural polymers. The proposed treatment can respond to the cell wall-degrading enzymes secreted by PWNs and vector insects during pine tree infestation to intelligently release pesticides to cut off the transmission and infestation pathways and realize the integrated control of PWN disease. The LC50 value of AVM@EC@Pectin was 11.19 mg/L for PWN and 26.31 mg/L for JPS. The insecticidal activity of AVM@EC@Pectin was higher than that of the commercial emulsifiable concentrate (AVM-EC), and the photostability, adhesion, and target penetration were improved. The half-life (t1/2) of AVM@EC@Pectin was 133.7 min, which is approximately twice that of AVM-EC (68.2 min). Sprayed and injected applications showed that nanopesticides had superior bidirectional transportation, with five-times higher AVM contents detected in the roots relative to those of AVM-EC when sprayed at the top. The safety experiment showed that the proposed treatment had lower toxicity and higher safety for nontarget organisms in the application environment and human cells. This study presents a green, safe, and effective strategy for the integrated management of PWN disease.

Biocontrol potential of entomopathogenic nematodes against the grey maize weevil Tanymecus dilaticollis (Coleoptera: Curculionidae) adults.

The grey maize weevil, Tanymecus dilaticollis, is a polyphagous species, which is among the most important pests of maize in Southeastern Europe. The efficacy of commercial products with two species of entomopathogenic nematodes (EPNs), Steinernema carpocapsae and Heterorhabditis bacteriophora, was investigated against adults of the grey maize weevil under laboratory conditions. Nemastar®, containing S. carpocapsae was more effective on T. dilaticollis adults than Nematop® containing H. bacteriophora, when applied uniformly to the surface of the soil, on Petri dishes containing T. dilaticollis adults. Results showed that S. carpocapsae rates of 83-333 infective juveniles/adult caused > 94% mortality in T. dilaticollis adults, whereas H. bacteriophora caused 27-61%, adult mortality, after exposure of insects to the commercial products of EPNs for 15 days. The infection rates of EPNs increased with concentration applied and ranged from 70-83% and 19-64% for Nemastar® and Nematop®, respectively. Subsequent field and semi-field tests were conducted with Nemastar® (application rate of 50 million S. carpocapsae per 100 m2) in maize crops with biological (mycoinsecticide Naturalis®, biofungicides and fertilizers) and chemical seed treatment (Gaucho® FS 600; active ingredient: imidacloprid) in Knezha, Bulgaria. Nematodes were found only in the dead specimens, in open plots and cages sprayed with the commercial nematode product. Nematode sprayings contributed for higher maize yields in the open maize plots in the fields with different seed treatments. We suggest that the use of powder formulation of S. carpocapsae in combination with biologically treated maize seeds can contribute to minimize the use of chemical insecticides against the grey maize weevil. The results obtained can be used as a base to further tests to ascertain the efficacy of EPNs products before they can be recommended for use in the integrated approach to T. dilaticollis management.

Identification of polyphenols from Rosa roxburghii Tratt pomace and evaluation of in vitro and in vivo antioxidant activity.

Rosa roxburghii Tratt pomace (RRTP) has increasingly attracted attention due to its various nutritional ingredients and health benefits. In this study, the free phenolic fraction (RRTP-FPF) and bound phenolic fraction (RRTP-BPF) were extracted from RRTP by solvent extraction method and alkaline hydrolysis method, respectively. The composition of polyphenols in RRTP-FPF and RRTP-BPF were identified by ultra-high performance liquid chromatography equipped with an electrospray ionization and quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS/MS). In vitro antioxidant assays indicated that RRTP-FPF and RRTP-BPF could scavenge radicals in a dose-dependent manner, and RRTP-BPF exhibited better scavenging activity than RRTP-FPF. In addition, RRTP-FPF and RRTP-BPF (20 âˆ¼ 100 µg/mL) treatment for 24 h could significantly increase the survival rate and decrease reactive oxygen species (ROS) level of paraquat-exposed nematodes through improving the activities of superoxide dismutase (SOD) and catalase (CAT). These results suggest that RRTP could be as a good and cheap source of natural antioxidants.

The effects of plant cysteine proteinases on the nematode cuticle.

BACKGROUND: Plant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The nematode cuticle is composed of collagens and cuticlins, but the specific molecular target(s) for the proteinases have yet to be identified. METHODS: This study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murine GI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms. RESULTS: Immunohistochemistry indicated that DPY-7 collagen is a target for CPs on the cuticle of C. elegans. The time course of loss of DPY-7 from the cuticle allowed us to use it to visualise the process of cuticle disruption. There was a marked difference in the time course of damage to the cuticles of the two species of nematode, with H. bakeri being more rapidly hydrolysed. In general, the CPs' mode of attack on the nematode cuticle was by degrading the structural proteins, leading to loss of integrity of the cuticle, and finally death of the nematode. Proteomic analysis failed conclusively to identify structural targets for CPs, but preliminary data suggested that COL-87 and CUT-19 may be important targets for the CPs, the digestion of which may contribute to cuticle disruption and death of the worm. Cuticle globin was also identified as a cuticular target. The presence of more than one target protein may slow the development of resistance against this new class of anthelmintic. CONCLUSIONS: Scanning electron microscopy and immunohistochemistry allowed the process of disruption of the cuticle to be followed with time. Cuticle collagens and cuticlins are molecular targets for plant cysteine proteinases. However, the presence of tyrosine cross-links in nematode cuticle proteins seriously impeded protein identification by proteomic analyses. Multiple cuticle targets exist, probably making resistance to this new anthelmintic slow to develop.

Chitosan and EDTA conjugated graphene oxide antinematodes in Eggplant: Toward improving plant immune response.

A new strategy regarding the fabrication of chitosan (CS) or ethylene diamine tetraacetic acid (EDTA) on graphene oxide (GO) was performed. The nematocidal potential against Meloidogyne incognita causing root-knot infection in eggplant was tested. The plant immune response was investigated through measuring the photosynthetic pigments, phenols and proline contents, oxidative stress, and antioxidant enzymes activity. Results indicating that, the treatment by pure GO recorded the most mortality percentages of M. incognita 2nd juveniles followed by GO-CS then GO-EDTA. In vivo greenhouse experiments reveals that, the most potent treatment in reducing nematodes was GO-CS which recorded 85.42%, 75.3%, 55.5%, 87.81%, and 81.32% in numbers of 2nd juveniles, galls, females, egg masses and the developmental stage, respectively. The highest chlorophyll a (104%), chlorophyll b (46%), total phenols (137.5%), and free proline (145.2%) were recorded in GO-CS. The highest malondialdehyde (MDA) value was achieved by GO-EDTA (7.22%), and hydrogen peroxide (H2O2) content by 47.51% after the treatment with pure GO. Treatment with GO-CS increased the activities of catalase (CAT) by 98.3%, peroxidase (POD) by 97.52%, polyphenol oxidase (PPO) by 113.8%, and superoxide dismutase (SOD) by 42.43%. The synthesized nanocomposites increases not only the nematocidal activity but also the plant systematic immune response.

Deciphering the synergistic impact of elevated temperature and oil pollution on meiobenthic community structure: A benthocosm study.

Meiobenthos has been considered as an excellent tool for biomonitoring assessment. Elevated temperature and oil pollution are considered as the most pervasive aspects of global environmental changes and matter of concern for contemporary society. Presently, very limited information is available about the synergistic effect of these stressors on meiobenthic community structure and tolerance potential from tropical intertidal environment. Here, we assessed their impacts on meiobenthic community by conducting a 60 days long benthocosm experiment selecting three sets of temperature (25°, 30° and 35 °C) and two sets of diesel oil (low and high) combinations. Gradual changes in their community composition were revealed discernibly with exposures to both the disturbances after 30 and 60 days of experimental period. Diversity profiles for the nematodes were less affected, but copepods showed a graded response of decreasing density with increasing dose of both the stressors. Other meiobenthic taxa such as halacarid mite, turbellaria and polycheate juveniles were adversely affected and eliminated from the treatments, howbeit abundance of ostracods, foraminiferans and bivalve settlers varied significantly. A 3-factor PERMANOVA indicated a significant effect of temperature, diesel, between their interaction and interaction among stressors and time on meiofaunal abundances. In case of free-living nematodes, temperature rise and diesel contamination synchronously led to an elimination of k-selected species like Halalaimus gracilis, H. longicaudatus, Oxystomina aesetosa and Pomponema sp. with a significant decrease in abundance of H. capitulatus and Oncholaimus sp. The r-selected species Daptonema invagiferoum, Sabatieria praedatrix, Theristus acer, Monhystera sp. and Thalassomonhystera sp. had endured even at high doses of diesel treatment in three different temperatures set up. The effects were evident in term of changes in life strategies with an increment of opportunistic species and increased trophic diversity of deposit feeders in treated sediments. Overall, elevated temperature together with diesel oil contamination were found to alter species dynamics within shallow intertidal meiobenthic communities, which might have significant Armageddon on benthic ecosystem functioning.

Ivermectin: An Anthelmintic, an Insecticide, and Much More.

Here we tell the story of ivermectin, describing its anthelmintic and insecticidal actions and recent studies that have sought to reposition ivermectin for the treatment of other diseases that are not caused by helminth and insect parasites. The standard theory of its anthelmintic and insecticidal mode of action is that it is a selective positive allosteric modulator of glutamate-gated chloride channels found in nematodes and insects. At higher concentrations, ivermectin also acts as an allosteric modulator of ion channels found in host central nervous systems. In addition, in tissue culture, at concentrations higher than anthelmintic concentrations, ivermectin shows antiviral, antimalarial, antimetabolic, and anticancer effects. Caution is required before extrapolating from these preliminary repositioning experiments to clinical use, particularly for Covid-19 treatment, because of the high concentrations of ivermectin used in tissue-culture experiments.

In vitro anthelmintic activity of Lippia gracilis Schauer essential oil against egg-hatching of goat gastrointestinal nematodes / Atividade anti-helmíntica in vitro do óleo essencial de Lippia gracilis Schauer contra a eclosão de nematódeos gastrintestinais de caprinos

Since drug-resistant nematodes became a common problem in sheep and goat industries, alternative methods using natural products have emerged as a viable and sustainable anthelmintic treatment option. Here, the in vitro effect of essential oil extracted from Lippia gracilis Schauer was assessed on the hatching process of nematodes recovered from naturally infected goats. Essential oil at concentrations of 0.08% (0.008 µL/mL), 0.12% (0.012 µL/mL), and 0.16% (0.016 µL/mL) was able to induce an average inhibition of 74.7, 84 and 93%, respectively. The effective concentration required to inhibit egg hatching in 50% of eggs (EC50) was 0.03452%. Therefore, essential oil of L. gracilis showed promisor in vitro anthelmintic results against egg-hatching of goat gastrointestinal nematodes.(AU)

Como os nematoides resistentes a drogas se tornaram um problema comum nas indústrias de ovinos e caprinos, métodos alternativos que utilizam produtos naturais surgiram como uma opção de tratamento anti-helmíntico viável e sustentável. Aqui, o efeito in vitro do óleo essencial extraído de Lippia gracilis Schauer foi avaliado no processo de eclosão de nematoides recuperados de caprinos naturalmente infectadas. O óleo essencial nas concentrações de 0,08% (0,008 µL/mL), 0,12% (0,012 µL/mL), e 0,16% (0,016 µL/mL)foi capaz de induzir uma inibição média de 74,7, 84 e 93%, respectivamente. A concentração efetiva necessária para inibir a eclosão de ovos em 50% dos ovos (CE50) foi de 0,03452%. Portanto, o óleo essencial de L. gracilis apresentou resultados anti-helmínticos in vitro promissores contra a eclosão de nematódeos gastrintestinais de caprinos.(AU)

Ovicidal and larvicidal potential of Rosmarinus officinalis to control gastrointestinal nematodes of sheep / Potencial ovicida e larvicida de Rosmarinus officinalis para controle de nematódeos gastrintestinais de ovinos

Abstract Gastrointestinal Nematode Infection (GIN) are the main constraint to the production of small ruminants. Studies of medicinal plants have been an important alternative in the effort to control these parasites. Therefore, the purpose of this study was to evaluate the in vitro ovicidal and larvicidal activity of essential oil of Rosmarinus officinalis. The oil was extracted, analyzed by gas chromatography and tested on GIN eggs and larvae in six concentrations, 227.5mg/mL, 113.7mg/mL, 56.8mg/mL, 28.4mg/mL, 14.2mg/mL and 7.1mg/mL. To determine the ovicidal activity, GIN eggs were recovered from sheep feces and incubated for 48h with different concentrations of the oil. For the evaluation of larval migration, third-stage larvae (L3) were obtained by fecal culture, and associated with the essential oil for 24h at the same concentrations, after which they were left for another 24 hours on microsieves, followed by the count of migrating and non-migrating larvae. The assays of R. officinalis oil showed a significant (p<0.05) 97.4% to 100% inhibition of egg hatching and a significant (p<0.05) 20% to 74% inhibition of larval migration. The main constituent revealed by gas chromatography was Eucalyptol. The results indicate that R. officinalis essential oil has ovicidal and larvicidal activity on sheep GINs.

Resumo As infecções por nematódeos gastrintestinais (ING) constituem a maior limitação à produção de pequenos ruminantes. Na busca do controle desses parasitos, estudos com plantas medicinais têm sido uma importante alternativa. Visto isto, o estudo desenvolvido teve como objetivo avaliar a ação ovicida e larvicida in vitro do óleo essencial de Rosmarinus officinalis. O óleo foi extraído, analisado por cromatografia gasosa e testado sobre ovos e larvas de ING em seis concentrações, 227,5mg/mL; 113,7mg/mL; 56,8mg/mL; 28,4mg/mL; 14,2mg/mL; 7,1mg/mL. Para determinar a ação ovicida, ovos de ING foram recuperados de fezes de ovinos e incubados por 48h com as diferentes concentrações do óleo. Na avaliação da migração das larvas, as larvas de terceiro estágio (L3) foram obtidas por coprocultura, e associadas ao óleo essencial por 24h nas mesmas concentrações, permanecendo por mais 24h em microtamises, seguindo-se a contagem de larvas que migraram e que não migraram. Os testes in vitro com o óleo de R. officinalis mostraram o nível de significância (p<0.05) 97,4% a 100% na inibição da eclodibilidade e 20% a 74% na inibição da migração das larvas. Na análise por cromatografia gasosa o constituinte majoritário foi o eucaliptol. Os resultados apresentados mostram que o óleo essencial de R. officinalis possui ação ovicida e larvicida sobre ING de ovinos.

Implication of the fecal egg count reduction test (FECRT) in sheep for better use of available drugs / Implicação do teste de redução da contagem de ovos nas fezes (TRCOF) em ovinos para melhor uso dos anti-helmínticos disponíveis

Abstract The aim here is to present data on the efficacy of anthelmintics in sheep flocks in Rio de Janeiro, Brazil, and to discuss the interpretation of the fecal egg count reduction test (FECRT) for each nematode genus. Fecal eggs counts and pre- and post-treatment coprocultures were performed, the former to evaluate the efficacy of and the latter to determine the overall parasite prevalence. An additional efficacy test was performed at Farm # 1 a year after the initial test. Severe anthelmintic resistance was found for the flocks, with no FECRT sensitivity at any of the 22 farms evaluated. However, an analysis of the infective larvae showed that some drugs were effective against certain parasitic genera; i.e., levamisole was more effective against Haemonchus spp. and moxidectin against Trichostrongylus spp. In the additional FECRT performed at Farm # 1, moxidectin and nitroxynil were ineffective separately, but when applied in combination they were highly effective due to their efficacy against Haemonchus (nitroxynil) and Trichostrongylus (moxidectin), respectively. The use of the FECRT targeting the parasitic nematode species prevalent on farms may make it possible to choose more effective anthelmintics.

Resumo O objetivo deste trabalho foi apresentar dados sobre a eficácia de anti-helmínticos em rebanhos ovinos no Rio de Janeiro, Brasil, e discutir a interpretação do teste de redução da contagem de ovos nas fezes (TRCOF) para cada gênero de nematoide. A contagem de ovos fecais (OPG) e coprocultura pré e pós-tratamento foram realizadas para avaliar a eficácia e a prevalência geral do parasito, respectivamente. Um teste de eficácia adicional foi realizado na Fazenda # 1 após um ano do teste inicial. Resistência anti-helmíntica grave foi encontrada, não havendo sensibilidade no TRCOF em nenhuma das 22 fazendas avaliadas. No entanto, na análise das larvas infectantes observou-se que algumas drogas foram eficazes contra certos gêneros parasitários; por exemplo, o levamisol foi mais eficaz contra Haemonchus spp. e a moxidectina contra Trichostrongylus spp. No TRCOF adicional realizado na Fazenda 1, a moxidectina e o nitroxinil foram ineficazes separadamente, mas quando aplicados em combinação, foram altamente eficazes devido à sua eficácia contra Haemonchus spp. (nitroxinil) e Trichostrongylus spp. (moxidectina), respectivamente. O TRCOF visando às espécies de nematoides parasitas prevalentes nas fazendas pode possibilitar a escolha de anti-helmínticos mais eficazes.

Anthelmintic-like activity of polyphenolic compounds and their interactions against the cattle nematode Cooperia punctata.

Polyphenolic compounds (PCs) have been proposed as one of the most bioactive group of secondary metabolites occurring in nature and have been associated to anthelmintic (AH)-like activity of plants against cattle nematodes. However, little is known regarding their synergetic / antagonistic interactions. This study assessed the in vitro AH-like activity of commercial PCs: quercetin, caffeic acid, rutin and coumarin, and their combinations against the egg hatching and larval exsheathment of Cooperia punctata; one of the most prevalent nematodes affecting grazing cattle in tropical regions. The molecules selected for the in vitro analysis were identified as bioactive phytochemicals of plants through bio-guided fractionation in previous studies. To estimate mean effective concentrations (EC50) five increasing concentrations were used for both Egg hatching inhibition assay (EHIA) and larval exsheathment inhibition assay (LEIA) (0.6-9.8 mg mL-1 and 0.15-2.4 mg mL-1, respectively). From the four molecules, only rutin did not affect egg hatching; while quercetin, showed no bioactivity against eggs or larvae (P > 0.766 and P > 0.621, respectively). Best-fit EC50 estimated through the EHIA was considered for PCs classification as bioactive (coumarin and caffeic acid) and non-bioactive (quercetin and rutin). Phytochemical interactions were subsequently assessed combining bioactive:non-bioactive PCs (8:2 ratio), and the nature of their interaction was classified using the fractional inhibitory concentration index (FICindex). Combinations had a highly synergistic interaction against larval exsheathment (FICindex < 0.5) except for coumarin:rutin against egg hatching (FICindex> 0.5). Quercetin and rutin acted as PCs AH-like activity enhancers, reducing EC50 of bioactive molecules in a range of 43%-64% and 68%-83% for EHIA and LEIA, respectively. A linear relationship between low molecular weight of molecules and ovicidal activity was observed; where, molecules with lower molecular weight displayed better-fit EC50 for ovicidal activity. Furthermore, coumarin and caffeic acid bioactivity against free-living stages of C. punctata makes them suitable candidates as markers for anthelmintic-like activity in bioactive forages. Combinations used through this investigation showed a potent anthelmintic-like activity against free-living forms of C. punctata, representing a first step towards the identification of promising alternatives for nematode control.

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.

Crop residues activity against the free-living stages of small ruminant nematodes / Atividade de resíduos de culturas contra os estágios de vida livre de nematoides de pequenos ruminantes

Abstract The nematicidal effect of different organic materials was evaluated in order to develop a non-chemical alternative soil treatment for control of the free-living stages of small ruminant gastrointestinal nematodes. The selected organic materials were residues from the juice industry of acerola, cashew, grape, guava, papaya and pineapple, as well as castor residue from the biodiesel industry. LC90 results showed that pineapple residue was the most efficient inhibitor of larval development, followed by castor, grape, cashew, acerola, guava and papaya. Castor residue was also a good source of nitrogen and was used in a greenhouse experiment to prevent larval development in contaminated goat faeces that was deposited in pots containing the grasses Brachiaria brizantha (var. Paiaguás) or Megathyrsus maximus x M. infestum (var. Massai). Castor residue caused a significant (P < 0.05) reduction (85.04%) in Paiaguás grass contamination (L3.dry mass-1) and a reduction of 17.35% in Massai grass contamination (P > 0.05), with an increase in the biomass production of Massai (251.43%, P < 0.05) and Paiaguás (109.19%, P > 0.05) grasses. This strategy, called Econemat®, with good results in vitro shows to be promising on pasture increasing phytomass production.

Resumo O efeito nematicida de diferentes materiais orgânicos foi avaliado, com o objetivo de desenvolver um tratamento alternativo não químico do solo para o controle dos estágios de vida livre de nematoides gastrintestinais de pequenos ruminantes. Os materiais orgânicos selecionados foram resíduos da indústria de suco de acerola, caju, uva, goiaba, mamão e abacaxi, além do resíduo de mamona da indústria de biodiesel. Os resultados da LC90 mostraram que o resíduo de abacaxi foi o mais eficiente inibidor do desenvolvimento larval, seguido pela mamona, uva, caju, acerola, goiaba e mamão. O resíduo de mamona também se mostrou uma boa fonte de nitrogênio, sendo usado em experimento em casa de vegetação para prevenir o desenvolvimento larval em fezes contaminadas, depositadas em vasos, contendo as gramíneas Brachiaria brizantha (var. Paiaguás) ou Megathyrsus maximus x M. infestum (var. Massai). O resíduo de mamona reduziu (85,04%) significativamente (P < 0,05) a contaminação no capim-Paiaguás (L3.dry massa-1), 17,35% no capim Massai (P> 0,05), e aumentou a produção de biomassa das gramíneas Massai (251,43%, P <0,05) e Paiaguás (109,19%, P> 0,05). Essa estratégia, chamada Econemat®, com bons resultados in vitro, mostra-se promissora nas pastagens aumentando a produção de fitomassa.

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.

Identification of small molecule enzyme inhibitors as broad-spectrum anthelmintics.

Targeting chokepoint enzymes in metabolic pathways has led to new drugs for cancers, autoimmune disorders and infectious diseases. This is also a cornerstone approach for discovery and development of anthelmintics against nematode and flatworm parasites. Here, we performed omics-driven knowledge-based identification of chokepoint enzymes as anthelmintic targets. We prioritized 10 of 186 phylogenetically conserved chokepoint enzymes and undertook a target class repurposing approach to test and identify new small molecules with broad spectrum anthelmintic activity. First, we identified and tested 94 commercially available compounds using an in vitro phenotypic assay, and discovered 11 hits that inhibited nematode motility. Based on these findings, we performed chemogenomic screening and tested 32 additional compounds, identifying 6 more active hits. Overall, 6 intestinal (single-species), 5 potential pan-intestinal (whipworm and hookworm) and 6 pan-Phylum Nematoda (intestinal and filarial species) small molecule inhibitors were identified, including multiple azoles, Tadalafil and Torin-1. The active hit compounds targeted three different target classes in humans, which are involved in various pathways, including carbohydrate, amino acid and nucleotide metabolism. Last, using representative inhibitors from each target class, we demonstrated in vivo efficacy characterized by negative effects on parasite fecundity in hamsters infected with hookworms.

Effects of the water-soluble fraction of a crude oil on freshwater meiofauna and nematode assemblages.

Oil water-soluble fractions (WSFs) compounds have low persistence in water; evaporating in a matter of hours to days. Nonetheless, they pose high toxicity to exposed biota. Their effects may be instantaneous or delayed, affecting, respectively, mainly survival vs growth and reproduction. We investigated the effects of crude oil WSFs on freshwater meiobenthos, with a focus on nematode assemblages, in community microcosm experiments lasting 15 weeks. Treatments consisted of the application of different concentrations of oil WSFs, i.e. high (100%) medium (50%) and low (10%), and effects were assessed one, three, nine and 15 weeks after contamination, allowing us to detect both short-term and lasting effects of oil-WSF. Additionally, we compared the effects of a single contamination event with those of a so-called 'constant' oil-WSF contamination where we replenished evaporated water with water containing the medium concentration of oil WSF. Next to nematodes, the most abundant meiofaunal taxa were rotifers, gastrotrichs, oligochaetes and tardigrades. Total abundance, different diversity indices, the composition of feeding-types and the age structure were investigated in the assessment of direct oil effects on the structure of nematode assemblages. Limited immediate effects were observed, except for a significant decrease of the index of taxonomic distinctness, which already appeared in the first week. Significant impacts on total nematode abundance, diversity and species composition only became apparent after 9-15 weeks of incubation, indicating that delayed effects of a single exposure are far more pronounced than instantaneous effects. Moreover, for most response variables, the strongest impacts were not observed in the highest-concentration treatment, but in a medium-concentration treatment with regular replenishment of oil WSF, suggesting that internal exposure may be important in generating effects. Furthermore, the predictability of the sensitivity of individual species was sometimes poor, which may not only result from these species' sensitivities, but also from alterations in interspecific interactions in polluted communities. Further toxicity tests should be carried out in order to unravel the main modes of action of crude oil WSF which lead to the observed long-term sublethal effects on nematode communities.

Nicotine-sensitive acetylcholine receptors are relevant pharmacological targets for the control of multidrug resistant parasitic nematodes.

The control of parasitic nematodes impacting animal health relies on the use of broad spectrum anthelmintics. However, intensive use of these drugs has led to the selection of resistant parasites in livestock industry. In that respect, there is currently an urgent need for novel compounds able to control resistant parasites. Nicotine has also historically been used as a de-wormer but was removed from the market when modern anthelmintics became available. The pharmacological target of nicotine has been identified in nematodes as acetylcholine-gated ion channels. Nicotinic-sensitive acetylcholine receptors (N-AChRs) therefore represent validated pharmacological targets that remain largely under-exploited. In the present study, using an automated larval migration assay (ALMA), we report that nicotinic derivatives efficiently paralyzed a multiple (benzimidazoles/levamisole/pyrantel/ivermectin) resistant field isolate of H. contortus. Using C. elegans as a model we confirmed that N-AChRs are preferential targets for nornicotine and anabasine. Functional expression of the homomeric N-AChR from C. elegans and the distantly related horse parasite Parascaris equorum in Xenopus oocytes highlighted some striking differences in their respective pharmacological properties towards nicotine derivative sensitivity. This work validates the exploitation of the nicotine receptors of parasitic nematodes as targets for the development of resistance-breaking compounds.

Nematode resistance to five anthelmintic classes in naturally infected sheep herds in Northeastern Brazil / Resistência de nematódeos a cinco classes de anti-helmínticos em rebanhos de ovinos naturalmente infectados no Nordeste do Brasil

Abstract This study aimed to evaluate the occurrence of nematode resistance to anthelmintics on sheep herds from the semi-arid region of Paraíba, Northeastern Brazil. Fecal Egg Count Reduction Test (FECRT) was carried out on 20 properties. In each herd, 30 animals were divided into five groups containing six animals each: group I, treated with albendazole 10%, 4 mg/kg; group II, ivermectin 0.08%, 0.2 mg/kg; group III, closantel 10%, 10 mg/kg; group IV, levamisole hydrochloride 5%, 5 mg/kg; and group V, monepantel 2.5%, 2.5 mg/kg. All treatments were administered orally as a single dose. Fecal samples were collected on days zero and 10 after treatment, to perform FECRT and coprocultures. Multiresistance was observed in all the properties; 95% had high resistance to albendazole, 85% to ivermectin, 80% to closantel, 40% to levamisole, and 45% to monepantel. On property 15, where monepantel was ineffective, a second FECRT for this anthelmintic was carried out 4 months after the first, resulting in 75% efficacy. Immediately after the FECRT result, two ewes were euthanized and necropsied, and Haemonchus contortus, Trichostrongylus axei, Trichostrongylus colubriformis, Oesophagostomum columbianum, and Trichuris ovis were recovered. It was concluded that the resistance of sheep gastrointestinal nematodes to antthelmintic, including monepantel, is high.

Resumo O objetivo deste estudo foi avaliar a ocorrência de resistência de nematódeos a anti-helmínticos em rebanhos ovinos do semiárido da Paraíba, Nordeste do Brasil. Testes de Redução na Contagem de Ovos Fecais (TRCOF) foram conduzidos em 20 propriedades. Em cada rebanho, 30 animais foram selecionados e foram divididos em cinco grupos contendo seis animais cada: grupo I, tratado com albendazol 10%, 10 mg/kg; grupo II, ivermectina 0,08%, 0,2 mg/kg; grupo III, closantel 10%, 10 mg/kg; grupo IV, cloridrato de levamisol 5%, 5 mg/kg; grupo V, monepantel 2,5%, 2,5 mg/kg. Todos os tratamentos foram administrados via oral, em dose única. Amostras fecais foram coletadas nos dias zero e 10 após os tratamentos, para a realização do TRCOF e coproculturas. Multirresistência foi observada em todas as propriedades; 95% dos rebanhos obtiveram alta resistência ao albendazole, 85% a ivermectina, 80% ao closantel, 40% ao levamisole, e 45% a monepantel. Na propriedade 15, onde o monepantel foi ineficaz, um segundo TRCOF foi conduzido para este fármaco 4 meses após o primeiro, resultando em 75% de eficácia. Imediatamente após o resultado do TRCOF, duas ovelhas foram eutanaziadas e necropsiadas, e Haemonchus contortus, Trichostrongylus axei, Trichostrongylus colubriformis, Oesophagostomum columbianum, e Trichuris ovis foram recuperados. Concluiu-se que a resistência dos nematódeos gastrintestinais de ovinos aos anti-helmínticos, incluindo o monepantel, é alta.

A randomized, blinded, controlled, multi-centered field study assessing the treatment of gastrointestinal nematode infections in cats with fluralaner plus moxidectin spot-on solution (Bravecto® Plus).

BACKGROUND: A spot-on formulation containing fluralaner (280 mg/ml) plus moxidectin (14 mg/ml) (Bravecto® Plus) was developed for the treatment of nematode infections as well as providing 12 weeks of protection against insect and acarine parasites in cats. The effectiveness and safety of this product against feline gastrointestinal nematodes was assessed in naturally-infested, client-owned cats under field conditions in Albania, Bulgaria, Germany and Hungary. METHODS: To be eligible for enrollment in this investigator-blinded study cats had to be at least 10 weeks-old, weigh at least 1.2 kg, be clinically healthy, and have a faecal sample testing positive for nematodes no more than eight days prior to treatment. Cats were stratified into blocks of three in order of presentation at each center and randomly allocated in a 2:1 ratio to be treated topically on Day 0 with fluralaner plus moxidectin (minimum dose rates 40 mg/kg and 2 mg/kg, respectively) or emodepside plus praziquantel (minimum dose rates 3 mg/kg and 12 mg/kg, respectively) (Profender®). Faecal samples were collected from cats prior to treatment and 14 ± 4 days later. RESULTS: There were 182 cats randomized to the fluralaner plus moxidectin group, and 91 to the emodepside plus praziquantel group. Prior to treatment the most commonly identified nematode egg was Toxocara cati, found in 79.1 and 82.4% of cats in the fluralaner plus moxidectin and emodepside plus praziquantel groups, respectively. Eggs of Toxascaris leonina were found in 8.2 and 6.6% of cats; of hookworms in 30.8 and 24.2%; and of Capillaria spp. in 7.1 and 4.3%, respectively. After treatment, faecal samples from 98.3% of fluralaner plus moxidectin treated and 96.6% of emodepside plus praziquantel-treated cats were free of nematode ova. Geometric mean faecal egg count reductions for T. cati, the only eggs found in post-treatment faecal samples, were 99.97% and 99.93%, respectively. Treatment with fluralaner plus moxidectin was non-inferior to emodepside plus praziquantel. Both products were safe and well tolerated by cats treated under field conditions. CONCLUSIONS: This field study confirms that, in addition to 12-week extended duration flea and tick control, fluralaner plus moxidectin provides broad spectrum treatment of nematodes in cats.

Nematicidal Cyclic Lipodepsipeptides and a Xanthocillin Derivative from a Phaeosphariaceous Fungus Parasitizing Eggs of the Plant Parasitic Nematode Heterodera filipjevi.

The new cyclic lipodepsipeptide ophiotine (1), two new arthrichitin derivatives named arthrichitins B (4) and C (5), a new xanthocillin-like alkaloid, xanthomide Z (2), and the previously described arthrichitin (3) were isolated from the liquid culture broth of a nematode-associated fungus with affinities to the genus Ophiosphaerella. The structural elucidation and determination of the absolute configuration of the new molecules were accomplished using a combination of spectroscopic and chemical techniques, including 1D and 2D NMR, HRMS, and Marfey's analysis. Opiotine (1) displayed moderate nematicidal activity against the host nematode ( Heterodera filipjevi), while xanthomide Z (2) exhibited very weak activity. Arthrichitin C (5) showed very weak cytotoxic effects on several cancer cell lines, with IC50 values in the range of 24-33 µM. Xanthomide Z is among few xanthocillin derivatives that comprise formamide functions instead of the cyano functions that are usually observed in this class of fungal alkaloids.