Multipurpose peptides: The venoms of Amazonian stinging ants contain anthelmintic ponericins with diverse predatory and defensive activities.

In the face of increasing drug resistance, the development of new anthelmintics is critical for controlling nematodes that parasitise livestock. Although hymenopteran venom toxins have attracted attention for applications in agriculture and medicine, few studies have explored their potential as anthelmintics. Here we assessed hymenopteran venoms as a possible source of new anthelmintic compounds by screening a panel of ten hymenopteran venoms against Haemonchus contortus, a major pathogenic nematode of ruminants. Using bioassay-guided fractionation coupled with liquid chromatography-tandem mass spectrometry, we identified four novel anthelmintic peptides (ponericins) from the venom of the neotropical ant Neoponera commutata and the previously described ponericin M-PONTX-Na1b from Neoponera apicalis venom. These peptides inhibit H. contortus development with IC50 values of 2.8-5.6 µM. Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α-helical conformations in lipid mimetic environments. We show that the ponericins induce non-specific membrane perturbation, which confers broad-spectrum antimicrobial, insecticidal, cytotoxic, hemolytic, and algogenic activities, with activity across all assays typically correlated. We also show for the first time that ponericins induce spontaneous pain behaviour when injected in mice. We propose that the broad-spectrum activity of the ponericins enables them to play both a predatory and defensive role in neoponeran ants, consistent with their high abundance in venom. This study reveals a broader functionality for ponericins than previously assumed, and highlights both the opportunities and challenges in pursuing ant venom peptides as potential therapeutics.

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.

Effects of different extracts of three Annona species on egg-hatching processes of Haemonchus contortus.

This study assessed the in vitro anthelmintic (AH) activity of methanol and acetone:water leaf extracts from Annona squamosa, A. muricata and A. reticulata against Haemonchus contortus eggs. The egg hatch test was used to determine the effective concentrations required to inhibit 50% of eggs hatching (EC50). The role of polyphenols on AH activity was measured through bioassays with and without polyvinylpolypyrrolidone (PVPP). Methanolic extracts mainly caused the death of eggs at the morula stage (ovicidal activity). Meanwhile, acetone:water extracts caused egg-hatching failure of developed larvae (larvae failing eclosion (LFE) activity). The lowest EC50 values against H. contortus eggs were observed for the methanolic extracts from A. reticulata and A. muricata (274.2 and 382.9 µg/ml, respectively). From the six extracts evaluated, the methanolic extracts of A. muricata, A. reticulata and A. squamosa showed the highest ovicidal activity, resulting in 98.9%, 92.8% and 95.1% egg mortality, respectively. When the methanolic extract of A. squamosa was incubated with PVPP, its AH activity increased. Similarly, when acetone:water extracts of A. muriata and A. reticulata were incubated with PVPP, their LFE activity increased. Alkaloids were only evident in methanolic extracts, irrespective of PVPP incubation. The presence of acetogenins was not observed. In conclusion, methanolic extracts obtained from leaves of A. muricata, A. reticulata and A. squamosa showed ovicidal activity affecting the morula of H. contortus eggs, with minor LFE activity. Meanwhile, acetone:water extracts showed mostly LFE activity, with a lower proportion of ovicidal activity.

In vitro predatory activity of nematophagous fungi isolated from water buffalo feces and from soil in the Mexican southeastern / Atividade predatória in vitro de fungos nematófagos isolados de fezes de búfalos e do solo no sudeste mexicano

Abstract Nematophagous fungi from the feces of water buffalo and soil from southeastern Mexico were isolated, and their in vitro predatory activity against Haemonchus contortus infective larvae (L3) (HcL3) was assessed. The fungi were isolated by sprinkling soil or feces on water agar plates. Six series of 10 Petri dishes containing a 7-day-old culture of each fungus and a series without fungi as the control were prepared. Five hundred HcL3 were added to each plate. The plates were incubated at room temperature. The average of recovered HcL3 was considered to estimate the larval reduction rate. Four nematophagous fungi isolates corresponding to Arthrobotrys oligospora, var microspora (strains 4-276, 269 and 50-80) and one identified as A. oligospora,var. oligospora (isolates 48-80) were obtained from water buffalo feces. From the soil, five isolates were isolated; three corresponded to A. musiformis (Bajío, Yumca and Macuspana isolates), and two isolates were identified as A. oligospora (Comalcalco and Jalapa de Méndez isolates). The predatory activity of isolates from water buffalo feces ranged between 85.9 and 100%. Meanwhile, the fungi from the soil ranged between 55.5 and 100% (p≤0.05). The nematophagous fungi obtained could have important implications in the control of parasites of importance in the livestock industry.

Resumo Fungos nematófagos das fezes de búfalo de água e do solo no sudeste do México foram isolados, e a atividade predatória in vitro contra larvas infectantes de Haemonchus contortus (L3) (HcL3) foi avaliada.Os fungos foram isolados por aspersão de solo e de fezes em placas de agar água. Foram preparadas seis séries de 10 placas de Petri contendo uma cultura de 7 dias de idade de cada fungo e uma série sem fungos como controle. Quinhentos HcL3 foram adicionadas a cada placa. As placas foram incubadas à temperatura ambiente. O número médio de HcL3 recuperadas foi considerado para estimar a taxa de redução larval. Quatro isolados de fungos nematófagos corresponderam a Arthrobotrys oligospora, var microspora (estirpes 4-276, 269 e 50-80) e um isolado identificado como A. oligospora, var. oligospora (isolados 48-80 de fezes de búfalo de água. Do solo, dos cinco isolados três corresponderam a A. musiformis (Bajío, Yumca e Macuspana isolados), e dois isolados foram identificados como A. oligospora (isolados de Comalcalco e Jalapa de Méndez). A atividade predatória de isolados de fezes de búfalo de água variou entre 85,9 e 100%. Enquanto isso, os fungos do solo variaram entre 55,5 e 100% (p≤0,05). Os fungos nematófagos obtidos podem ter importantes implicações nesse controle de parasitos de importância na indústria pecuária.

Evaluation of antioxidant and oxidant status of goats (Capra aegagrus hircus) naturally infected with Haemonchus contortus.

The present study aimed to assess the antioxidant and oxidant status of goats naturally infected with Haemonchus contortus. Based upon the parasite burden, infection in goats was categorized as heavy (> 500 worms), mild (100-500 worms) or low (< 100 worms). Abomasal tissues from non-infected and infected goats were used for the determination of catalase (CAT), glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), aspartate (AST) and alanine (ALT) aminotransferases, acid (ACP) and alkaline (ALP) phosphatases, superoxide content (O2-), protein carbonyl (PC), malondialdehyde (MDA) and reduced glutathione (GSH). A significantly higher level of CAT, GST and GR activity and a lower level of GPx activity were recorded in infected compared to non-infected tissue. A significant increase in the level of AST, ALT, ALP and ACP was found in the abomasal tissue of the infected animals, which was related to the worm burden. The oxidative stress markers were also altered, with a significant decline in GSH levels, whereas MDA, PC and O2- concentrations showed a marked increase. In conclusion, it has been demonstrated that haemonchosis in goats resulted in considerable oxidative stress, which was directly related to the worm burden.

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.

The effect of ovine peripheral blood mononuclear cells on Haemonchus contortus larval morbidity in vitro.

The objective of this study was to determine the effects of peripheral blood mononuclear cells (PBMC), derived from parasite-resistant St. Croix (STC) hair sheep and parasite-susceptible Suffolk (SUF) sheep, on Haemonchus contortus L3 stage larval death in vitro, with or without autologous serum. Larval morbidity was quantified by measuring larval ATP concentration following incubation with PBMC. Larvae exposed to either STC- or SUF-derived PBMC had lower ATP than live larvae (0.12 µmol/L ATP and 0.16 µmol/L ATP vs 0.27 µmol/L ATP, respectively) (P<.001) and greater ATP of dead larvae (0.03 µmol/L ATP) (P<.001). Breed differences were observed with addition of autologous serum. Larvae exposed to SUF-derived PBMC with autologous serum were not significantly different from live larval ATP. STC-derived serum did not significantly reduce larval ATP compared to PBMC alone (0.11 µmol/L ATP), but was significantly reduced compared to live larvae (0.22 µmol/L ATP) and SUF-derived PBMC with autologous serum (0.23 µmol/L ATP) (P<.001). These data indicate that a cellular response alone is capable of significantly reducing larval ATP in a breed-independent manner. However, addition of serum to SUF-PBMC failed to reduce larval ATP, indicating breed-dependent humoral response to H. contortus.

In vitro and in vivo studies of the native isolates of nematophagous fungi from China against the larvae of trichostrongylides.

To screen potential nematophagous fungi candidates for the biological control of parasitic nematodes in livestock, in vitro and in vivo studies of the native isolates of nematophagous fungi against the larvae of trichostrongylides were conducted. The in vitro predatory activity of 16 native nematophagous fungal isolates on the larvae of trichostrongylides in sheep feces was assessed. In the ten isolates of Duddingtonia flagrans, the reduction percentage for the infective larvae (L3) of Trichostrongylus colubriformis ranged from 57.21 to 99.83%, and that of Haemonchus contortus ranged from 62.12 to 99.88%. The analysis of the same assay on five isolates of Arthrobotrys superba and one isolate of A. cookedickinson (Monacrosporium cystosporum) showed comparable results with those for D. flagrans. To determine the excretion time of fungal isolates in feces after oral administration, D. flagrans (SDH035) were studied in vivo in sheep and rabbits. Results showed that the tested fungal isolates existed in sheep feces from 12 to 72 h after fungal treatment, and the fungal excretion in rabbit feces occurred at 4 h, reached a peak at 10 h, and declined gradually 18 h after oral administration. All the native fungal isolates were assessed after passing through the gastrointestinal tract of sheep. Treatment with isolates of D. flagrans significantly reduced the number of developing larvae in the feces, and the efficacies ranged from 55.15 to 98.82%. One out of the five isolates of A. superba and A. cookedickinson (BS002) survived after passing through the gastrointestinal tract, and the L3 reduction rates were 83.79 and 81.33%, respectively. Results of the present study provide information about the in vitro predatory activity of nematophagous fungi from China on the L3 of trichostrongylides and their ability to pass through the gastrointestinal tract before administering them for biocontrol.

Early IL-4 gene expression in abomasum is associated with resistance to Haemonchus contortus in hair and wool sheep breeds.

Early immune events associated with reduced larval burden remain unclear in parasite-resistant breeds of sheep. Therefore, our objective was to determine breed differences in immune-related gene expression following infection with H. contortus. Gene expression in abomasal tissue and mucosa and in abomasal lymph nodes (ALN) was measured in 24 St. Croix (hair) lambs and 24 Dorset x (Finn-Rambouillet) (wool) lambs at 0 (uninfected), 3, 5 and 7 days after infection with 10 000 L3 H. contortus larvae. Expression of IL-4 in abomasal mucosa was detected on day 3 and increased to day 7 in hair lambs, but was not detectable in wool lambs. Genes that recruit neutrophils (CXCL1) and macrophages (MCP1) were upregulated in abomasal mucosa of hair lambs. Genes associated with alternative macrophage activation (ARG-1) and eosinophil activation (Gal-14) were also upregulated in the abomasal mucosa of hair lambs. Tissue remodeling genes (MMP13, PDGF) and tumour necrosis factor alpha (TNF-α) and MCP1 were upregulated in abomasal tissue of wool lambs; these lambs also had greater expression of forkhead box P3 in ALN. These data indicate a role for early IL-4 expression locally and demonstrate potential downregulation of immunity in wool sheep that could facilitate establishment of H. contortus.

Chemical Structures of Plant Hydrolyzable Tannins Reveal Their in Vitro Activity against Egg Hatching and Motility of Haemonchus contortus Nematodes.

The use of synthetic drugs against gastrointestinal nematodes of ruminants has led to a situation where resistance to anthelmintics is widespread, and there is an urgent need for alternative solutions for parasite control. One promising approach is to use polyphenol-rich bioactive plants in animal feeds as natural anthelmintics. In the present work, the in vitro activity of a series of 33 hydrolyzable tannins (HTs) and their hydrolysis product, gallic acid, against egg hatching and motility of L1 and L2 stage Haemonchus contortus larvae was studied. The effect of the selected compounds on egg and larval structure was further studied by scanning electron microscopy. The results indicated clear relationships between HT structure and anthelmintic activity. While HT size, overall flexibility, the types and numbers of functional groups, together with the linkage types between monomeric HTs affected the activity differently, the optimal structure was found with pentagalloylglucose.

Relative expression analysis of IL-5 and IL-6 genes in tropical sheep breed Pelibuey infected with Haemonchus contortus.

Haemonchus contortus is a parasitic nematode of Pelibuey sheep, a meat breed used in tropical regions. Due to anthelmintic problems, the identification of hosts resistant to H. contortus is another option of control. The aim of this study was to analyse the relative expression of IL-5 and IL-6 genes in Pelibuey sheep after H. contortus infection. Nineteen lambs infected with H. contortus and three more lambs without infection were studied. The haemonchosis was determined by the number of eggs per gram of faeces (epg) and by the estimation of the percentage of the packed cell volume (%pcv). Peripheral blood mononuclear cells (PBMCs) were obtained to extract RNA at 0, 1, 2, 7, 14, 21 and 28 days after infection to quantify the relative expression of IL-5, IL-6 and GAPDH by real-time PCR. Five lambs were classified as low responders (lr) to haemonchosis with averages of 1519 ± 315·3 epg and 31·49 ± 5·13%pcv, and 14 lambs were identified as high responders (hr) with averages of 530 ± 132 epg and 34·88 ± 3·75%pcv. The expression ratio of IL-5 was significantly different compared with control lambs at 2, 7 and 14 days post-infection (PI), and IL-6 was significantly different after 14 days. The highest level of relative expression for IL-5 and IL-6 genes was 9·9-fold and 12-fold after 2 and 14 days for hr hosts (P < 0·05) compared with control group, respectively. In conclusion, the Pelibuey breed in grazing areas exhibited different expression of IL-5 and IL-6 obtained from PBMCs against H. contortus, suggesting the importance of these cytokines in regulating the nematode infection.

In vitro ovicidal and larvicidal activity of the essential oil of Artemisia lancea against Haemonchus contortus (Strongylida).

Prolonged use of chemical anthelmintics has been found to result in anthelmintic resistance and environmental issues, thereby limiting the application of these drugs in domestic animals and prompting interest in the study of plant extracts as alternative sources thereof. The aim of this study was to evaluate the in vitro effect of the essential oil (EO) of Artemisia lancea against the parasitic nematode Haemonchus contortus using egg hatch assay, larval development assay, and larval migration inhibition assay. The EO yield of extraction was 0.63% (w/w), and the major constituents were 1,8-cineole (34.56%) and camphor (16.65%). In the egg hatch assay, an inhibition greater than 99% was observed with the EO at 10 mg mL(-1) and the LC50 was 1.82 mg mL(-1). 1,8-Cineole demonstrated moderate ovicidal activity with a LC50 of 4.64 mg mL(-1), whereas camphor did not show enough activity to have its LC50 determined. In the larval development assay, the EO, 1,8-cineole, and camphor inhibited 93.6%, 65.2%, and 57% of larval development at 10 mg mL(-1) and exhibited dose-dependent responses with LC50 values of 1.66, 5.07, and 7.80 mg mL(-1), respectively. In the migration inhibition assay, the EO and 1,8-cineole at best inhibited 77% and 60.3% of larval migration at 10 mg mL(-1), respectively. Camphor showed low inhibition capacity, and its efficacy was not dose dependent. The results indicate that the in vitro anthelmintic activity of the EO of A. lancea may be associated with the additive action of the two major constituents, as well as other more minor terpenoid components.

Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep.

Sheep have a variable ability to resist gastrointestinal nematode infection, but the key factors mediating this response are poorly defined. Here we report the first large-scale application of quantitative proteomic technologies to define proteins that are differentially abundant between sheep selectively bred to have an enhanced (resistant) or reduced (susceptible) ability to eliminate nematodes. Samples were collected from the abomasal mucosa three days after experimental challenge with the nematode, Haemonchus contortus. This timing reflects the initial interaction of host and parasite, and the tissue represents the immediate interface. We identified and quantified more than 4400 unique proteins, of which 158 proteins showed >1.5 fold difference between the resistant and susceptible sheep. Trefoil factor 2, a member of RAS oncogene family (RAP1A) and ring finger protein 126 were amongst the proteins found to be highly abundant in the abomasal surface of resistant sheep, whereas adenosine deaminase and the gastrokine-3 like precursor were found at higher levels in susceptible sheep. Construction of gut proteome interaction networks identified mitochondrial function and energetic partitioning as important components of an effective nematode eliminating response. The differentially abundant proteins may be useful targets for phenotypic tests that aim to identify sheep with an enhanced ability to resist nematode infection.

The kinetics of local cytokine and galectin expression after challenge infection with the gastrointestinal nematode, Haemonchus contortus.

Gastrointestinal nematode parasites undergo several developmental stages within their mammalian host, each presenting different antigenic challenges to the immune system. To examine the expression of different immune mediators over time, biopsy samples were collected from the cannulated abomasum (true stomach) of immune sheep at several times after a challenge infection with Haemonchus contortus L3s. IL-5 and IL-13 mRNA expression levels were significantly increased above saline-challenged control levels at 5 and 7 days post challenge, while IL-4 showed an earlier peak at day 2 post challenge. IL-5, IL-13 and IL-4, as well as IFN-γ mRNA levels, peaked at 7 days before decreasing to non-significant levels at 28 days post challenge. TNF-α followed a similar profile while there was a slight increase in TGF-ß in both control and challenged sheep. There was a significant increase in galectin-14 mRNA in the L3 challenged compared with the saline challenged group at 7 days while both galectin-11 protein and mRNA levels increased significantly by day 3 post challenge, peaking at 5-7 days post challenge. Distinct correlations were observed between these immune parameters at different times after L3 challenge. The galectin-14 protein level at day 2 post challenge was the only measured mediator significantly negatively correlated with worm burden. These studies highlight the dynamic nature of the immune response during parasite infection and the need to consider the different life cycle stages involved.

Cyclotide interactions with the nematode external surface.

Cyclotides are a large family of cyclic cystine knot-containing plant peptides that have anthelminthic activities against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematodes of sheep. In this study, we investigated the interaction of the prototypic cyclotide kalata B1 with the external surface of H. contortus larvae and adult worms. We show that cyclotides do not need to be ingested by the worms to exert their toxic effects but that an interaction with the external surface alone is toxic. Evidence for this was the toxicity toward adult worms in the presence of a chemically induced pharyngeal ligature and toxicity of cyclotides toward nonfeeding larval life stages. Uptake of tritiated inulin in ligated adult worms was increased in the presence of cyclotide, suggesting that cyclotides increase the permeability of the external membranes of adult nematodes. Polyethylene glycols of various sizes showed protective effects on the nonfeeding larval life stage, as well as in hemolytic activity assays, suggesting that discrete pores are formed in the membrane surfaces by cyclotides and that these can be blocked by polyethylene glycols of appropriate size. This increased permeability is consistent with recently reported effects of cyclotides on membranes in which kalata B1 was demonstrated to form pores and cause leakage of vesicle/cellular contents. Our data, together with known size constraints on the movement of permeants across nematode cuticle layers, suggest that one action of the cyclotides involves an interaction with the lipid-rich epicuticle layer at the surface of the worm.

[Ovicidal and larvicidal activity in vitro of Eucalyptus globulus essential oils on Haemonchus contortus]. / Atividade ovicida e larvicida in vitro do óleo essencial de Eucalyptus globulus sobre Haemonchus contortus.

The objective of this work was to evaluate ovicidal and larvicidal effects of Eucalyptus globulus essential oil (EGEO) on Haemonchus contortus. The chemical composition determination of EGEO was through gas chromatography and mass spectrometry. Egg hatch test (EHT) was performed in concentrations 21.75; 17.4; 8.7; 5.43 e 2.71 mg x mL(-1). In larval development test (LDT) were used the concentrations 43.5; 21.75; 10.87; 5.43 e 2.71 mg x mL(-1). Each trial was conducted by negative control with Tween 80 (3%) and positive control, 0.02 mg x mL(-1) of thiabendazole in EHT and 0.008 mg x mL(-1) of ivermectin in LDT. The maximum effectiveness of EGEO on eggs was 99.3% in concentration of 21.75 mg x mL(-1) and on larvae was 98.7% in concentration 43.5 mg x mL(-1). The concentration of EGEO that inhibits 50% of the eggs and larvae was 8.3 and 6.92 mg x mL(-1), respectively. The oil chemical analysis identified as main component the monoterpen 1,8-cineol. EGEO presented ovicidal and larvicidal activities in vitro, revealing a good potential for use in the control of sheep and goat gastrointestinal nematodes.

Atividade ovicida e larvicida in vitro do óleo essencial de Eucalyptus globulus sobre Haemonchus contortus / Ovicidal and larvicidal activity in vitro of Eucalyptus globulus essential oils on Haemonchus contortus

O objetivo deste trabalho foi avaliar o efeito do óleo essencial de Eucalyptus globulus (OEEG) sobre a eclosão e desenvolvimento de larvas de Haemonchus contortus. A determinação da composição química do OEEG foi feita por meio de cromatografia gasosa acoplada à espectrometria de massa. No teste de eclosão de ovos (TEO), utilizaram-se as concentrações 21,75; 17,4; 8,7; 5,43 e 2,71 mg.mL-1; e no de desenvolvimento larvar (TDL) as concentrações foram 43,5; 21,75; 10,87; 5,43 e 2,71 mg.mL-1. Cada ensaio foi acompanhado por um controle positivo, 0,02 mg.mL-1 tiabendazol, no TEO e 0,008 mg.mL-1 ivermectina no TDL, e por um controle negativo com o diluente Tween 80 (3 por cento). A eficácia máxima obtida pelo OEEG sobre ovos foi de 99,3 por cento na concentração de 21,75 mg.mL-1 e, sobre larvas, 98,7 por cento na concentração de 43,5 mg.mL-1. A concentração do OEEG, que inviabilizou 50 por cento dos ovos e das larvas do parasito, foi de 8,3 e 6,92 mg.mL-1, respectivamente. A análise química do óleo identificou como principal componente o monoterpeno 1,8-cineol. O OEEG apresentou atividade ovicida e larvicida in vitro sobre H. contortus, determinando um bom potencial para utilização no controle de nematóides gastrintestinais de ovinos e caprinos.

The objective of this work was to evaluate ovicidal and larvicidal effects of Eucalyptus globulus essential oil (EGEO) on Haemonchus contortus. The chemical composition determination of EGEO was through gas chromatography and mass spectrometry. Egg hatch test (EHT) was performed in concentrations 21.75; 17.4; 8.7; 5.43 e 2.71 mg.mL-1. In larval development test (LDT) were used the concentrations 43.5; 21.75; 10.87; 5.43 e 2.71 mg.mL-1. Each trial was conducted by negative control with Tween 80 (3 percent) and positive control, 0.02 mg.mL -1 of thiabendazole in EHT and 0.008 mg.mL-1 of ivermectin in LDT. The maximum effectiveness of EGEO on eggs was 99.3 percent in concentration of 21.75 mg.mL-1 and on larvae was 98.7 percent in concentration 43.5 mg.mL-1. The concentration of EGEO that inhibits 50 percent of the eggs and larvae was 8.3 and 6.92 mg.mL-1, respectively. The oil chemical analysis identified as main component the monoterpen 1,8-cineol. EGEO presented ovicidal and larvicidal activities in vitro, revealing a good potential for use in the control of sheep and goat gastrointestinal nematodes.

Haemonchus contortus calreticulin binds to C-reactive protein of its host, a novel survival strategy of the parasite.

Calreticulin (CalR), a Ca(2+) binding multifunctional protein, is secreted by the parasitic nematode Haemonchus contortus. We have earlier observed binding of this protein to a 24-kDa polypeptide (p24) present in an enriched preparation of prothrombin. In the present study, the identity of p24 was established as a C-reactive protein (CRP) by several criteria. CalR binding to CRP is an elegant strategy devised by the parasite to survive in the host. The secreted CalR may achieve this either by limiting the free concentration of CRP, which has antiparasite activity or inhibit the activation of the classical complement pathway triggered on binding of CRP to C1q protein. CalR binding to CRP would also ensure a check on the procoagulant activity of the CRP enabling parasite to feed on the host blood. Thus, targeting CalR could be a novel strategy to tackle this parasite, which has developed resistance to many anthelmintics.

Cellular phenotypes in the abomasal mucosa and abomasal lymph nodes of goats infected with Haemonchus contortus.

The distribution of T-cell subsets (CD2, CD4, CD8, and gammadelta) and B cells (IgM) was examined at 3, 6, 10 and 13 days post-infection (dpi) in the abomasal mucosa and abomasal lymph nodes of goats primarily infected with Haemonchus contortus. In the abomasal mucosa a mild (3 and 6 dpi) or marked (10 and 13 dpi) increase of T cells, particularly CD4+ and gammadelta+ lymphocytes, was observed, whereas the increase in CD8+ cells was less pronounced. B cells and IgG+ plasma cells also showed a marked increase in the abomasal mucosa at 10 and 13 dpi. The abomasal lymph nodes showed an increase in size, particularly at 10 and 13 dpi, and a decrease in the proportion of T cells, particularly CD8+ lymphocytes, due to the increased proportion of B cells. The proportion of CD4+ and gammadelta+ lymphocytes did not change significantly during the infection in the abomasal lymph nodes, but their absolute numbers were augmented as a result of the enlargement of the nodes. The results revealed a strong cellular and humoral immune response during the early post-infection stages. However, as indicated by the worm burdens, this rapid host response was unable to induce larval expulsion.

Characterization of Haemonchus contortus calreticulin suggests its role in feeding and immune evasion by the parasite.

Haemonchus contortus, a gastrointestinal parasite of sheep and goat feeds on the blood of its host and causes bleeding at the biting site. In this report, we demonstrate that the Ca2+ binding protein, calreticulin (CalR), is present in excretory/secretory products of adult worms. The secreted CalR enhanced plasma coagulation time. Using recombinant fragments, this property has been mapped to C-terminal part of the molecule which has binding sites for Ca2+ as well as clotting factors. Complement protein C1q bound to immobilized CalR and C1q dependent lysis of sensitized sheep erythrocytes was inhibited by CalR, a function mapped to N-domain of the protein. Factor X and a 24 kDa polypeptide derived from prothrombin but not prothrombin bound to immobilized CalR. The binding site for 24 kDa polypeptide in the CalR molecule has been localized in the P-domain. Our results suggest at least two functions for secreted CalR: first, to prevent blood clotting by binding to Ca2+ and clotting factors thus enabling parasite to feed on the host blood and second to modulate the host immune response by binding to complement C1q thereby facilitating parasite's survival within the host.