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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

Thermotaxis and thermosensory neurons in infective larvae of Haemonchus contortus, a passively ingested nematode parasite.

As a basis for studies of thermal behavior of infective larvae (L3) of Haemonchus contortus resulting from ablation of amphidial neurons, the locations of the amphidial cell bodies in the hatchling larva (L1) were compared with their locations in the L3. We sought to verify that killing each targeted cell body in L1 destroys the putative corresponding dendrite of the L3. These comparisons confirmed the predicted cell body-to-dendrite connections, as well as similarities in the general amphidial structure of the two stages. We then conducted a series of studies using laser microbeam ablation of amphidial cell bodies in the L1 to determine the role of specific neurons in the thermal behavior of the L3. In a thermal gradient, normal L3 of H. contortus migrate to the temperature at which they were cultured and/or maintained. Larvae grown at 16 degrees or 26 degrees C migrate appropriately to either of these temperatures. Larvae grown to the L3 stage at 16 degrees C and then moved to 26 degrees C become acclimated to this temperature and thereafter migrate to it. However, when the putative thermosensory neurons, the finger cell neurons (AFD), were ablated in hatchling larvae with a laser microbeam, and these were grown to the L3 stage and tested on a radial thermal gradient, they failed to migrate to their culture temperature. Instead, they moved actively and continuously over much of the assay plate surface, with no obviously oriented cryo- or thermotactic movement. Ablation-control larvae, those in which putatively chemosensory neuron classes ASE or AWC were killed, migrated normally to their culture temperature. When the RIA interneurons (identified by positional homology with those of Caenorhabditis elegans) were ablated, the operated larvae moved actively, but circled near the initial placement point; control larvae, in which other nonamphidial neurons were killed, migrated normally. These results indicate that the finger cell neurons (AFD) are the primary thermosensory class in H. contortus. The RIA-class neurons integrate thermal responses in H. contortus, as do their putative structural homologs in C. elegans, but the behavior of H. contortus subsequent to RIA ablation is strikingly different.

Sensory neuroanatomy of a passively ingested nematode parasite, Haemonchus contortus: amphidial neurons of the first stage larva.

When infective larvae of Haemonchus contortus (a highly pathogenic, economically important, gastric parasite of ruminants) are ingested by grazing hosts, they are exposed to environmental changes in the rumen, which stimulate resumption of development. Presumably, resumption is controlled by sensory neurons in sensilla known as amphids. Neuronal function can be determined by ablation of specifically recognized neurons in hatchling larvae (L1) in which neuronal cell bodies are easily visualized using differential interference microscopy. Using three-dimensional reconstructions from electron micrographs of serial transverse sections, amphidial structure of the L1 is described. Each amphid of H. contortus is innervated by 12 neurons. The ciliated dendritic processes of 10 neurons lie in the amphidial channel. Three of these end in double processes, resulting in 13 sensory cilia in the channel. One process, that of the so-called finger cell, ends in a number of digitiform projections. Another specialized dendrite enters the amphidial channel, but leaves it to end within the sheath cell, a hollow, flask-shaped cell that forms the base of the amphidial channel. Although not flattened, this process is otherwise similar to the wing cells in Caenorhabditis elegans; we consider it AWC of this group. Two other neurons, ASA and ADB, appear to be homologs of wing cells AWA and AWB in C. elegans, although they end as ciliated processes in the amphidial channel, rather than as flattened endings seen in C. elegans. Each of the 12 amphidial neurons was traced to its cell body in the lateral ganglion, posterior to the worm's nerve ring. The positions of these bodies were similar to their counterparts in C. elegans; they were named accordingly. A map for identifying the amphidial cell bodies in the living L1 was prepared, so that laser microbeam ablation studies can be conducted. These will determine which neurons are involved in the infective process, as well as others important in establishing the host-parasite relationship.

Biochemical and immunochemical characterization of 125I-labeled cuticle components of Haemonchus contortus.

Live Haemonchus contortus developmental stages were radioiodinated and then subjected to a stepwise extraction procedure consisting of a buffer extract (with or without detergent) to solubilize putative surface-associated antigenic macromolecules, followed by a detergent/beta-mercaptoethanol (BME) extract to solubilize putative cuticle collagen proteins. A buffer-extracted iodinated 100-kDa protein was present in the free-living, infective L3(2M) stage. This labeled protein was released during in vitro exsheathment of L3(2M) and was not present in the ecdysed second molt (2M) cuticle. In addition to the 100-kDa protein, exsheathment fluid contained a 70-kDa labeled protein that was not extracted from iodinated L3(2M) with either detergent or BME. The data suggest that these proteins are components of the specialized ring portion of the 2M cuticle that is enzymatically ruptured during ecdysis. The L3(2M) and the exsheathed third-stage larvae (L3) contained 3 labeled, BME-extracted, collagenase-sensitive proteins of 108, 88 and 53 kDa. In contrast, four detergent-extracted, collagenase-insensitive, iodinated proteins (143, 81, 58 and 30 kDa) were present in adult H. contortus. The 143-kDa protein was both glycosylated and immunogenic. All 4 adult cuticle proteins were released from the cuticle surface into culture fluids. Furthermore, a cysteine protease was secreted by adults which apparently hydrolyzed the released 81-, 58- and 30-kDa surface proteins.

The effects of closantel treatment on the ultrastructure of Haemonchus contortus.

H. contortus were recovered from sheep 0-14 h after intramuscular treatment with closantel. Ultrastructural examination revealed that mitochondria were more electron dense and contained swollen cristae compared with untreated controls. Following treatment, the basal channels in the intestine became prominent and there was vesicle formation in all organs examined. In contrast, closantel-resistant H. contortus appeared normal after drug treatment. It is likely that closantel affects membrane associated processes responsible for fluid and ion homeostasis as well as mitochondrial function. Untreated H. contortus were maintained in balanced salt solution for 12 h which caused lesions indicative of fluid imbalance, but at 23 h there were serious structural abnormalities.

A helical, polymeric extracellular protein associated with the luminal surface of Haemonchus contortus intestinal cells.

The structure of the intestinal cells of the parasitic nematode Haemonchus contortus is described. The cells have numerous microvilli about 0-09 micron in diameter; most being 5-5-7-5 micron in length. The microvillar (plasma) membrane is coated with a layer of amorphous material (glycocalyx) about 60 A thick which is electron dense in sectioned preparations. Associated with the surface of this material, and filing the spaces between the microvilli, are filaments in the form of helices about 400 A in diameter and of variable pitch. The helices appear to be flexible but they are aligned approximately with the long axes of the microvilli. There are up to ten helices per microvillus; they extend beyond the tips of the microvilli and are up to 10 micron long. The material has been obtained nearly pure in small amounts. It is primarily protein and it is proposed that it should be called contortin. The monomeric form (of molecular weight about 60,000) has been identified with a Y-shaped structure with arms about 45 A long and 25 A wide seen in negatively stained preparations. The helical filament appears to be formed by lateral polymerization of patirs of these units.