Combination of synthetic anthelmintics and monoterpenes: Assessment of efficacy, and ultrastructural and biophysical properties of Haemonchus contortus using atomic force microscopy.

The resistance of Haemonchus contortus to synthetic anthelmintics is of increasing concern; and different strategies are being evaluated to improve parasite control. The present study investigated the in vitro effects of combinations of synthetic compounds and monoterpenes. Additionally, the chemical association of the best combinations and their impact on the ultrastructural and biophysical properties of H. contortus eggs was evaluated. We assessed the efficacy of the monoterpenes, carvacrol, thymol, r-carvone, s-carvone, citral, and p-cymene and the anthelmintics, albendazole and levamisole using the egg hatch test (EHT) and the larval migration inhibition test (LMIT), respectively. The minimum effective concentrations of the monoterpenes, according to the EHT (efficacy ranging from 4.4%-11.8%) and LMIT (efficacy ranging from 5.6%-7.4%), were used in combination with different concentrations of synthetic compounds, and the IC50 and synergism rate (SR) were calculated. Fourier-transform infrared spectroscopy (FTIR) was used to analyze the chemical association between the best combinations as revealed by the in vitro tests (albendazole and levamisole with r-carvone or s-carvone). Atomic force microscopy (AFM) was used to assess the ultrastructural and biophysical properties of H. contortus eggs treated with the albendazole and r-carvone combination. Among the monoterpenes, the highest efficacies were exhibited by carvacrol (IC50 = 185.9 µg/mL) and thymol (IC50 = 187.0 µg/mL), according to the EHT, and s-carvone and carvacrol (IC50 = 1526.0 and 1785.3 µg/mL, respectively), according to the LMIT. According to the EHT, albendazole showed a slight statistically significant synergism in combination with r-carvone (SR = 3.8) and s-carvone (SR = 3.0). According to the LMIT, among the monoterpenes, r-carvone (SR = 1.7) and s-carvone (SR = 1.7) showed an increase in efficacy with levamisole; however, this was not statistically significant. The FTIR spectra of albendazole and levamisole, in association with r-carvone and s-carvone, indicated the presence of chemical interactions between the synthetic and natural molecules, contributing to the possible synergistic effects of these associations. Eggs treated with albendazole and r-carvone showed an increase in roughness and a decrease in height, suggesting that the treatment induced damage to the egg surface and an overflow of its internal contents. Overall, the combination of albendazole with r-carvone and s-carvone was efficacious against H. contortus, demonstrating a chemical association between the compounds; the significant changes in the egg ultrastructure justify this efficacy.

In vitro effects of Moringa oleifera seed lectins on Haemonchus contortus in larval and adult stages.

Haemonchus contortus is a hematophagous parasite causing damage to the production of ruminant animals throughout the world. This study evaluated the in vitro effect of proteins from Moringa oleifera (WSMoL - Water Soluble M. oleifera Lectin and cMoL - coagulant M. oleifera Lectin) on the motility of infective larvae and adult male and female worms of H. contortus. The specific activity of total proteases and the morphology of the worms exposed to the lectins were observed. Both lectins inhibited motility of all parasite stages tested. WSMoL and cMoL at 500 µg mL-1 interfered in the motility of larvae. Values of 11.1% and 8.1% were the lowest motility indices of larvae with sheath, and 30.6% and 16.4% were the lowest motility indices of exsheathed larvae treated with WSMoL and cMoL, respectively. In 1 mg mL-1 solutions of WSMoL and of cMoL, the motility index of adult male worms was 23.3% (p < 0.001) and 20% (p < 0.001), while the motility index of adult female worms was 63.3% (p > 0.05) and 26.6% (p < 0.001), respectively. Greater proteolytic activity was detected in extracts obtained from adult worms, male and female, after incubation with the lectins. Morphological changes caused by the lectins were revealed by changes in the crests of the cuticle, in the longitudinal striations and at the vulva.

Cuminaldehyde induces oxidative stress-mediated physical damage and death of Haemonchus contortus.

Cuminaldehyde (CA), a monoterpenoid, preset in many plant sources including cumin, induces reactive oxygen-related damage and death in Haemonchus contortus, a parasitic worm with an LD50, values of 127.3 ±â€¯7.5, 184.5 ±â€¯12.1 and 104.1 ±â€¯7.9 µg/mL for an adult female, adult male worms (12 h) and L3 larvae, respectively (24 h). Fifty percent of inhibition of egg hatching (IC50) was obtained at 142.4 ±â€¯11.4 µg/mL after 48 h of exposure. Scanning electron microscopy revealed physical damage to the anterior and posterior ends, intestinal, ovarian, and esophageal regions of the warms on exposure to ca. The exposure of worms to CA also led to a systemic increase in reactive oxygen species (ROS) within 3 h. The better activity was seen with CA compared to standard antihelminthic drug albendazole (Alb). 74 µg/mL CA showed 2.3 fold more increase of catalase (CAT), 0.61 fold increase of superoxide dismutase (SOD), 3.3 fold increase of glutathione peroxidase (GPx) activity and 17.5 fold increase of glutathione (GSH) activity in comparison with Alb (500 µg/mL) for the same time of exposure (3 h). A firm increase of (2.9 fold) was also observed in nitric oxide synthase (NOS) activity within 12 h of exposure with CA (74 µg/mL) in comparison with Alb. Therefore the preclinical potential of CA is much higher than widely used antihelminthic drug Alb. The results open new opportunities to explore CA as a new active antihelminthic molecule.

Assessment of biophysical properties of Haemonchus contortus from different life cycle stages with atomic force microscopy.

Atomic force microscopy (AFM) was used in this work to investigate the ultrastructural and mechanical characteristics of Haemonchus contortus, the major gastrointestinal nematode that infects small ruminants worldwide. The biophysical characterization of this species is extremely important in order to reveal mechanisms of action of drugs and to classify its ultrastructure and biomechanical properties. High-resolution topographic images by AFM as well as data on biomechanical properties of cuticles were obtained at different stages of H. contortus. The results reveal details of the mechanical and structural properties of this nematode never observed before for nematodes parasite with other microscope techniques. Qualitative and quantitative reductions in the elasticity of the larvae stage egg were compared with those of the morulae stage, and the increased adhesion of unsheathed L3 were compared with the same stage of sheathed larvae. The results presented here open possibilities for understanding the mechanisms of drug and biomolecular actions that can be used to control infections caused by H. contortus.

Chemical constituents of Calotropis procera latex and ultrastructural effects on Haemonchus contortus / Constituintes químicos do látex de Calotropis procera e efeitos ultraestruturais em Haemonchus contortus

Abstract This study aimed to evaluate the anthelmintic and ultrastructural effects of Calotropis procera latex on Haemonchus contortus. C. procera latex was twice centrifuged at 10,000×g and dialyzed to obtain a fraction rich in proteins, named LP (latex protein), and at 3,000 rpm to obtain a fraction rich in secondary metabolites, named LNP (latex non-protein). Specimens of H. contortus exposed to LNP, LP and PBS in the Adult Worm Motility Test (AWMT) were submitted to scanning (SEM) and transmission (TEM) electron microscopy to verify changes in their ultrastructure. Phytochemical tests in the LNP indicated the presence of phenols, steroids, alkaloids and cardenolides. High-Performance Liquid Chromatography (HPLC) characterized the presence of the compounds gallic acid and quercetin in the LNP. The protein content in the LP was 43.1 ± 1.1 mg/mL and 7.7 ± 0.3 mg/mL in LNP. In AWMT, LNP and LP inhibited the motility of 100% of the nematodes, with LNP being more effective than LP and ivermectin more effective than both (p <0.05). Cuticle changes were observed by SEM and TEM in nematodes treated with LP and LNP. Calotropis procera latex has anthelmintic effects against H. contortus, causing damage to its cuticle and other alterations in its ultrastructure.

Resumo Este estudo objetivou avaliar os efeitos anti-helmínticos e ultraestruturais do látex de Calotropis procera sobre Haemonchus contortus. Látex de C. procera foi centrifugado duas vezes à a 10.000xg e dialisado para obter uma fração rica em proteínas, denominada proteínas do látex (LP). E centrifugado e centrifugado a 3.000 rpm, para obter uma fração rica em metabólitos secundários, denominada LNP (látex não proteico). Espécimes de H. contortus expostos à LNP, LP e PBS no Teste de Motilidade dos Nematoides Adultos (TMNA) foram submetidos a microscopia eletrônica de varredura (MEV) e de transmissão (MET), para verificar alterações em sua ultraestrutura. Testes fitoquímicos em LNP indicaram a presença de fenóis, esteroides, alcaloides e cardenolídeos. A presença dos compostos ácido gálico e quercetina em LNP foi caracterizada por Cromatografia Líquida de Alta Eficiência (CLAE). O conteúdo de proteínas em LP foi de 43,1 ± 1,1 mg/mL e de 7,7 ± 0,3 mg/mL em LNP. No TMNA, LNP e LP inibiram a motilidade de 100% dos nematoides, sendo LNP mais eficaz que LP, e a ivermectina mais eficaz que ambos (p <0,05). Alterações na cutícula de nematoides tratados com LP e LNP foram observadas por MEV e MET. O látex de C. procera apresenta efeito anti-helmíntico sobre H. contortus, causando danos à sua cutícula e outras alterações em sua ultraestrutura.

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.

Ultrastructural study of adult Haemonchus contortus exposed to polyphenol-rich materials under in vivo conditions in goats.

This study assessed the ultrastructural changes caused in adult Haemonchus contortus obtained from goats fed fodder based on polyphenol-rich plants Lysiloma latisiliquum or Onobrychis viciifolia or from goats drenched with quebracho extract, Schinopsis spp. The H. contortus were obtained from artificially infected goats used as models to investigate the anthelmintic effect of feeding or drenching with the polyphenol-rich materials. Nematode populations were exposed to polyphenol-rich plant materials by feeding host goats for 8 consecutive days (D28 to D35 post-infection) with (a) L. latisiliquum fodder at 800 g fresh basis/day, (b) O. viciifolia fodder offered ad libitum, and (c) drenched with a solution containing quebracho extract (90 g/day). Meanwhile, control H. contortus were obtained from goats fed polyphenol-free diets. The H. contortus specimens were recovered from the goats on D36 post-infection, and transmission electron microscopy (TEM) was used to identify ultrastructural changes. In vivo exposure to different polyphenol-rich plant materials caused vacuolization of the nematodes' intestinal, muscular and hypodermal cells. These alterations represent the first evidence of cell damage caused in H. contortus when hosts were fed or drenched with polyphenol-rich materials. Ultrastructural changes affecting several types of cells could explain modifications in worm motility and nutrition, eventually affecting H. contortus reproductive success. This study contributes to our understanding of the mechanisms of action of polyphenol-rich plants against H. contortus when given as nutraceuticals to goats.

TITLE: Étude ultrastructurale d'Haemonchus contortus adultes exposés in vivo à des matériaux riches en polyphénols chez les chèvres. ABSTRACT: Cette étude a examiné les lésions ultrastructurales provoquées chez les adultes d'Haemonchus contortus obtenus chez des chèvres alimentées par des fourrages riches en polyphénols, Lysiloma latisiliquum ou Onobrychis viciifolia, ou des chèvres recevant des extraits de quebracho, Schinopsis spp. Les H. contortus ont été obtenus à partir de chèvres artificiellement infestées pour vérifier les effets anthelminthiques de l'alimentation ou l'administration de ressources riches en polyphénols. Les populations de nématodes ont été exposées à des ressources végétales contenant des polyphénols chez des hôtes infestés recevant pendant 8 jours (J28 à J35 après infestation) : (a) un fourrage à base de L. latisiliquum (800 g/jour), (b) un fourrage à base de sainfoin (O. viciifolia) offert ad libitum, et (c) une solution d'extrait de quebracho (90 g/jour). Pendant ce temps, une population témoin d'H. contortus a été obtenue à partir de chèvres alimentées avec un régime à base de fourrages sans polyphénols. Des échantillons d'H. contortus ont été obtenus à partir des divers groupes de chèvres à J36 après infestation, et la microscopie électronique à transmission a été utilisée pour identifier les modifications ultrastructurales. L'exposition in vivo à différents matériaux végétaux riches en polyphénols chez l'hôte a provoqué chez les nématodes une vacuolisation des cellules intestinales, musculaires et hypodermiques. Ces altérations représentent la première preuve de lésions cellulaire provoquées chez H. contortus lorsque l'hôte reçoit des ressources riches en polyphénols. Les changements ultrastructuraux affectant divers types de cellules pourraient expliquer des modifications fonctionnelles de motilité et de nutrition des vers, et finalement modifier le succès reproducteur d'H. contortus. Cette étude contribue à la compréhension des mécanismes d'action des plantes contenant des polyphénols utilisées comme alicament contre H. contortus.

Ultrastructure changes induced by the phloroglucinol derivative agrimol G isolated from Leucosidea sericea in Haemonchus contortus.

Plant extracts used for the treatment of helminth infections in sheep are an alternative to chemical anthelmintic drugs. Previous studies have reported the anthelmintic activity of acetone leaf extracts of Leucosidea sericea. For this study, we evaluate the ultrastructure changes induced by the acetone leaf extract of L. sericea and the component agrimol G (AG) that was isolated for the first time on adult haemonchus parasites. Adult haemonchus parasites harvested from sheep were incubated with the plant extract and AG for 3 h and evaluated by both scanning and transmission electron microscopy in comparison and in combination with albendazole or ivermectin. In all cases the method of evaluation shows ultrastructural changes, with albendazole inducing mitochondrial damage and ivermectin inducing muscle degeneration, both as previously described. Incubation with the plant extract and AG resulted in the formation of numerous non-membrane bound multi-vesicular like bodies and evenly spread disruptions/erosion in the epicuticle. Combining AG with ivermectin or albendazole resulted in an absence of effect of AG. Based on the structural changes induced by AG, together with the absence of an effect in combination with ivermectin and albendazole would suggest a disrupted microtubular network. The latter does however require biochemical confirmation.

Anthelmintic effect and tissue alterations induced in vitro by hydrolysable tannins on the adult stage of the gastrointestinal nematode Haemonchus contortus.

Haemonchus contortus constitutes a severe problem for ruminant's production, it is the most frequent nematode parasite both in template and tropical regions, induces economical losses, and shows increasing resistance to currently available anthelmintics. Tannins are secondary metabolites that naturally fulfill defense functions in plants, representing a non-conventional, natural alternative in the treatment of gastrointestinal parasites in sheep. The objective of this work was to explore the in vitro anthelmintic activity of hydrolysable tannins on adult stage of Haemonchus contortus. Adults nematodes were obtained directly from the abomasum of ovines, and a dose response curve was performed with tannins extract at 0, 2, 4, 8, 25 and 50 mg/mL, and a time response curve at 0.5, 1, 2 and 24 h at 37 °C. Hydrolysable tannins decreased motility and induced mortality of H. contortus. We observed cuticle disruption around the mouth and reproductive organs, as well as evisceration. After 24 h of exposition, 8 mg/mL induced 83% of mortality and with 25 mg/kg 100% of mortality was achieved. The LD50 was 3.54 mg/mL, while LD90 was 10 mg/mL. We propose hydrolysable tannins as an alternative to contribute in the nematode control in ruminants.

In vitro ovicidal activity of Baccharis conferta Kunth against Haemonchus contortus.

The indiscriminate use of chemical drugs to deworm livestock tends to trigger an anthelmintic resistance problem. In this context, the use of plant extracts rich in secondary metabolites could be an alternative method for the control of gastrointestinal nematodes. Baccharis conferta Kunth is a native plant species from Mexico that is widely used by several ethnic groups as forage for farm animals and medicinally to treat gastrointestinal diseases such as acute stomach ache, dysentery, diarrhoea, vomiting, indigestion, colic, intestinal spasms, urinary problems, and cramps. The aim of the present study was to isolate and characterise the ovicidal constituents of B. conferta and to determine a possible mode of action against Haemonchus contortus. The ovicidal activity was determined using the egg hatching inhibition test (EHI) to assess the methanol extract obtained from B. conferta foliage. The dry extract was partitioned (water/ethyl acetate) to obtain an ethyl acetate (BcEtOAc-F) and aqueous fraction. BcEtOAc-F showed an ovicidal activity of 72.32% EHI at 1 mg/mL. The chromatographic fractionation of BcEtOAc-F resulted in three active sub-fractions with higher ovicidal activity: BcC1R4 (99.15% EHI at 1.0 mg/mL); BcC1R5 (92.51% EHI at 0.75 mg/mL); and BcC1R8 (96.8% EHI at 3.0 mg/mL). Chemical analysis of the BcC1R4 fraction allowed the identification of the major active compound, isokaempferide (1, 98.06% EHI at 1 mg/mL). While, 4,5-di-O-acid caffeoylquinic (3; 96.8% EHI at 3 mg/mL) and an inactive flavone (vicenin-2, 2) were identified as the main compounds in BcC1R8. Chemical characterisation of the isolated compounds was performed via spectroscopic (NMR) and spectrometric (UPLC-MS) analyses. Additionally, the environmental and confocal scanning microscopy analyses revealed that isokaempferide was able to cross the eggshell layer without breaking it and attach itself to the embryo, causing its death. The flavonol, isokaempferide, and the hydroxycinamic acid, 4,5-di-O-caffeoylquinic, displayed powerful ovicidal effects, proving to be a potential alternative for the development of a phytodrug for the control of haemonchosis.

Chemical composition and in vitro activity of Calotropis procera (Ait.) latex on Haemonchus contortus.

Calotropis procera is among the species of medicinal plants that have traditionally been used for the treatment of parasites in small ruminants, stimulating the scientific validation of anthelmintic effects. This study aimed to investigate the chemical composition of ethyl acetate extract of Calotropis procera latex (EAECPL), assess the in vitro effect against Haemonchus contortus and the structural changes caused in the adult worm. The latex was collected, lyophilized and subjected to washing with the ethyl acetate solvent to obtain EAECPL. The constituents of the extract were isolated by column chromatography and identified by (13)C and (1)H nuclear magnetic resonance spectroscopy. The egg hatching test (EHT), larval development test (LDT) and adult worms motility test (WMT) were conducted to evaluate the effectiveness of EAECPL on eggs, larvae and adult of H. contortus, respectively. The worms obtained from the WMT, after 24h exposure to EAECPL or controls were observed on a scanning electron microscope (SEM). The results were analysed by variance analysis and compared with Tukey's test (P<0.05). Three compounds were isolated from EAECPL and identified as urs-19(29)-en-3-yl acetate, (3ß)-Urs-19(29)-en-3-ol, and 1-(2',5'-dimethoxyphenyl)-glycerol. In the EHT, EAECPL inhibited larval hatching by 91.8% at dose of 4mg/ml. In the LDT 1mg/ml inhibited 99.8% larval development. In the WMT, EAECPL in the concentration of 100µg/ml inhibited 100% motility of worms, 12h post-exposition. In the SEM, obvious differences were not detected between the negative control worms and the worms treated with EAECPL. In this study, EAECPL showed an effect on inhibition egg hatching, larval development and motility of the adult worms of H. contortus. This should be related both to the identified compounds, as well as the other compounds present in the EAECPL, acting alone or synergistically.

Effect of sericea lespedeza leaf meal pellets on adult female Haemonchus contortus in goats.

Sericea lespedeza (SL; Lespedeza cuneata) is a perennial warm-season forage rich in condensed tannins (CT) that has been reported to have anthelmintic activity against small ruminant gastrointestinal nematodes (GIN), particularly Haemonchus contortus, a highly pathogenic blood-feeder, but the mechanism of action of CT against H. contortus is not clearly understood. An experiment with young goats was designed to study the effect of SL leaf meal pellets on (1) a mature H. contortus infection, and (2) the surface appearance of adult H. contortus female worms. Thirty-six female and castrated male Boer crossbred goats artificially infected with H. contortus larvae were fed 75% SL leaf meal pellets or alfalfa pellets (18 goats/treatment group) in a 28-day confinement feeding trial. Fecal and blood samples were collected weekly for fecal egg count (FEC) and packed cell volume (PCV) determination, respectively, and all goats were slaughtered at the end of the trial for adult GIN recovery and counting. Five adult female H. contortus were recovered from the abomasum of two goats from each treatment group and from a prior study in which 75% and 95% SL leaf meal pellets or a commercial feed pellet were group-fed to grazing goats (270 days old, Spanish males, 10/treatment group) at 0.91 kg/head/d for 11 weeks. Adult GIN collected were fixed and examined for evidence of surface damage using scanning electron microscopy. Feeding 75% SL pellets to young goats in confinement reduced (P<0.05) FEC compared with control animals, while total worm numbers and PCV were not influenced by treatment. Three out of the 5 adult H. contortus recovered from SL treatment goats in the confinement feeding trial had cuticular surface damage, while no damage was observed on worms from the control group. All five worms observed from both SL treatments in the grazing study showed a shrunken, disheveled cuticular surface, whereas this was not observed on worms from control animals. Overall, this work suggests that a possible mechanism of action of SL against female H. contortus in the animal's abomasum is a direct action of CT on the cuticle of the worm.

Haemonchus longistipes Railliet & Henry, 1909 (Nematoda, Trichostrongylidae) from the Egyptian dromedary, Camelus dromedarius (Artiodactyla: Camelidae), first identification on the basis of light and ultrastructural data.

Haemonchus longistipes is a gastrointestinal abomasal nematode which is one of the most prevalent and pathogenic parasites infesting the stomach of ruminants. On the basis of light and ultrastructural data, the objective of the present study was to introduce a first identification of the cameline haemonchosis caused by H. longistipes. Abomasa of 42 Egyptian camels Camelus dromedarius (Artiodactyla: Camelidae) were collected monthly from September 2013 to April 2014 from the main slaughter house of Cairo, Egypt. Adult male and female nematode worms were recovered from 26 (62%) specimens of the examined abomasa. The parasites were of yellow color; the body was filiform (slender) tapered towards the anterior end in male and towards both ends in female. Buccal capsules absent, the buccal cavity was small with a conspicuous dorsal lancet extended from dorsal wall. The cervical papillae were prominent and spine-like. The body length of the female worm was 16.6-20.5 (18.5 ± 0.3) mm. The anterior end to the cervical papillae was 3.19-4.30 (4.12 ± 0.5) mm. The vulva of the female had a linguiform process or flap, the tail is without a spine, and the anal pore at the posterior end of the body had a simple dorsal rim. The body of male was 10.4-14.7 (13.9 ± 2.0) mm in length. The male bursa had elongated lobes supported by long, slender rays. The small dorsal lobe was asymmetrical with Y-shaped dorsal rays. The spicules were long with a length of 0.52-0.54 (0.53 ± 0.05) mm, each provided with a small barb and pore near its extremity. Synlophe was bilaterally and dorsoventrally symmetrical; it extended from cephalic expansion over anterior 50% of prebursal or prevulvar body and consisted of a maximum of 42 ridges. The described species herein was compared with the three morphologically similar species Haemonchus mitchelli, Haemonchus okapiae, and H. longistipes with their synlophes consist of 42 ridges distributed over the anterior half of the body. These species can be separated by unique structural characteristics of their synlophes, spicules, and copulatory bursa. The most morphologically similar species to the recovered worm was H. longistipes. Also, some of the parameters with regard to morphology and morphometry of this parasite were described for the first time.

Scanning electron microscopy of Haemonchus contortus exposed to tannin-rich plants under in vivo and in vitro conditions.

The structural changes induced in adult Haemonchus contortus after in vitro and in vivo contact with a tannin-rich (TR) plant, either tzalam (Lysiloma latisiliquum) or sainfoin (Onobrychis viciifolia), were assessed using scanning electron microscopy (SEM). All the worms used in the study were adult females. The Haemonchus adult worms were obtained from the abomasum of infected donor goats. Adult H. contortus were kept in contact with the extracts of TR plants for 24h for the in vitro assays and were compared to worms maintained in PBS (control). For the in vivo studies, the adult H. contortus parasites were obtained from artificially infected goats. All the goats were fed a tannin-free diet until D27 post-infection when infection was patent. On D28 some goats were fed fodder of one of the TR plants for seven consecutive days. Thus, their H. contortus were in contact with TR fodder within the gastrointestinal tract. The control worms were obtained from goats fed only a tannin-free diet. In the in vitro assays and in vivo studies, the SEM observations revealed structural alterations in the worms after contact with TR plants when compared to the control worms (i.e.: longitudinal and transversal folds and thicker cuticular ridges, material aggregates around the buccal capsule and/or vulva or anus). The main changes concerned the cuticle and the buccal area. The structural changes found in the worms exposed to TR plants might affect their motility and nutrition with possible consequences on their reproduction, as suggested by previous in vivo trials.

Comparative analysis of destruction of the infective forms of Trichuris trichiura and Haemonchus contortus by nematophagous fungi Pochonia chlamydosporia; Duddingtonia flagrans and Monacrosporium thaumasium by scanning electron microscopy.

The present study aimed to demonstrate by scanning electron microscopy (SEM) the in vitro predatory activity of nematophagous fungi Pochonia chlamydosporia (VC1 and VC4 isolates) Duddingtonia flagrans (AC001 isolate) and Monacrosporium thaumasium (NF34a isolate) on eggs of Trichuristrichiura and infective larvae (L3) of Haemonchus contortus. The work was divided into two experimental tests (A and B). In tests A and B, the predatory activity of nematophagous fungi P. chlamydosporia, D. flagrans and M. thaumasium on eggs of T. trichiura and H. contortus L3 was observed. After 6h, in test A, isolates P. chlamydosporia (VC1 and VC4) had a role in destroying eggs of T.trichiura. For fungi D. flagrans and M. thaumasium the ovicidal activity on T. trichiura eggs was not observed. Test B showed that D. flagrans (AC001) and M. thaumasium (NF34a) were capable of predating H. contortus L3, but no predation by the fungus P. chlamydosporia was seen. These fungi can offer potential for the biological control of nematodes.

Interaction between the nematophagous fungus Duddingtonia flagrans and infective larvae of Haemonchus contortus (Nematoda: Trichostrongyloidea).

The interaction between Duddingtonia flagrans and infective larvae of Haemonchus contortus was studied in vitro under optical and scanning electron microscopy. Trap formation by the fungus started 9 hours after inoculation and first larvae were found 11 hours after larval inoculation on colonies grown on the surface of dialysis membranes. Scanning electron micrographs were taken 12, 24, 36 and 48 h after larval predation. Details of predation structures and fungus-larvae interaction are described. A mucilaginous substance occurred at the points of adherence of traps to nematode cuticle. Bacteria were also found at some points of interaction between fungus and larval cuticle. Cuticle penetration by fungus hyphae occurred only 48 h after predation.

Amphidial structure of ivermectin-resistant and susceptible laboratory and field strains of Haemonchus contortus.

The development of anthelmintic resistance by nematode parasites is a growing problem for veterinarians, pet owners, and producers. The intensive use of the macrocyclic lactones for the treatment of a variety of parasitic diseases has hastened the development of resistance to this family of parasiticides. As a result, resistance to ivermectin, moxidectin, nemadectin, and doramectin by Haemonchus contortus has been documented throughout the world. Sensory neurons located in the cephalic end of nematodes are in close contact with the external environment. Through these neurons, important chemical and thermal cues are gathered by the parasite. Examination of serial electron micrographs of ivermectin-susceptible and ivermectin-resistant H. contortus allows for comparison of neuronal structure, arrangement of neurons within the amphidial channel, and distance of the tip of the dendritic processes to the amphidial pore. The latter of these characteristics provides a useful means by which to compare the association between the neurons and the external environment of the worm. Comparison of parental laboratory strains of ivermectin-susceptible strains of H. contortus with related selected, ivermectin-resistant strains and with a wild-type ivermectin-susceptible field strain of H. contortus from Louisiana reveal that the ivermectin-resistant worms examined have markedly shorter sensory cilia than their ivermectin-susceptible parental counterparts. Additionally, the amphidial neurons of ivermectin-resistant worms are characterized by generalized degeneration and loss of detail, whereas other neurons outside of the channels, such as the labial and cephalic neurons, are normal in structure. These findings raise a number of questions regarding the relationship between amphidial structure and ivermectin resistance as well as the role of amphids as a means of entry for ivermectin. While shortened amphidial sensilla are associated with ivermectin resistance, it remains unclear if such a structural modification facilitates survival of nematodes exposed to macrocyclic lactones.

Redescriptions of Haemonchus mitchelli and Haemonchus okapiae (Nematoda: Trichostrongyloidea) and description of a unique synlophe for the haemonchinae.

In the course of a revision of Haemonchus Cobb, 1898 (Nematoda), commonly referred to as large stomach worms, significant new morphological information was discovered that allows the recognition of 2 species believed for more than 50 yr to be synonymous. Both species, Haemonchus mitchelli Le Roux, 1929, from the eland Taurotragus oryx and other African ruminants and H. okapiae van den Berghe, 1937, from the okapi Okapia johnstoni, have a synlophe of 42 ridges, but the synlophe of H. mitchelli is longer than that of H. okapiae. The distal tip of the left spicule of H. mitchelli bears a barb that is about twice as long as the short barb and half as long as the long barb on the right spicule. In contrast, the barb on the left spicule of H. okapiae is similar in size to the short barb and about 25% as long as the long barb of the right spicule. The dorsal ray of H. mitchelli is bifurcated distally for 25-39% (32%) of its length and its stem is expanded proximally, but the dorsal ray of H. okapiae is bifurcated 37-50% (42%) and its stem is of uniform thickness.

Localization of p-glycoprotein mRNA in the tissues of Haemonchus contortus adult worms and its relative abundance in drug-selected and susceptible strains.

Membrane transporter P-glycoproteins (Pgps) are present in a number of nematode species, including Haemonchus contortus. Allelic variation in some Pgp genes has been found to be associated with resistance to the anthelmintic ivermectin (IVM), although functional verification of a role for Pgps in IVM resistance has yet to be demonstrated. By in situ hybridization, the distribution of Pgp mRNA was visualized in transverse cryosections of adult H. contortus, using a digoxigenin-labeled cDNA probe encoding the ATP-binding region of an H. contortus Pgp. The probe sequence targeted a conserved ATP-binding region of Pgp-A (97.9% identity). It also shared 49.7-71.1% identity with 11 other Pgp sequences previously identified in H. contortus and may hybridize these sequences to give an overall measure of the total P-glycoprotein mRNA. Staining was predominately localized along the intestinal tract of the worms, with the most intense staining localized in the pharynx and anterior intestine. In the mid- and posterior intestinal regions, staining was restricted to the luminal side of the intestine. Some staining was also associated with the vas deferens and the lateral hypodermal chords anterior to the nerve ring. Using densitometry, the levels of Pgp mRNA in the pharynges of unselected and IVM- and moxidectin (MOX)-selected strains of male and female H. contortus were compared. No differences were detected between the levels of expression of Pgp in the susceptible strain versus the IVM- or MOX-selected strains. Evidence in the literature suggests that not all Pgp homologues are linked to chemical resistance phenotypes. It is thus possible that expression of I of the H. contortus Pgps is altered in IVM-resistant strains but that this phenomenon was undetectable in our experiments.