Effect of norepinephrine treatment on Haemonchus contortus and its excretory products

Haemonchus contortus is a highly pathogenic gastrointestinal nematode of small ruminant animals. In modern intensive farming, livestock often suffer from different types of stress. However, whether host stress hormones influence H. contortus infection is largely unknown. Therefore, we treated H. contortus with norepinephrine (NE) and analyzed the changes in its excretory/secretory products (ESPs). Label-free quantitative proteomic analysis was used to identify differences in body proteins and ESPs between the control and NE-treated groups. We also investigated the changes in ESP action by analyzing cytokine secretion and goat peripheral blood mononuclear cell (PBMC) proliferation after incubation with ESPs secreted by NE-treated H. contortus. Thirty-two proteins in the body samples and 137 in the ESPs were differentially expressed between the groups. Gene ontology (GO) annotation showed that the functions of these different proteins might be involved in energy metabolism, protein metabolism, lipid metabolism, redox homeostasis, ion channel, and cell structure. NE treatment caused oxidative stress in H. contortus and changed the expression levels of some immunogenic proteins, such as the 15-kDa ESP. Meanwhile, the ESPs secreted by NE-treated H. contortus significantly decreased PBMC proliferation and the interleukin (IL)-2, IL-4, and interferon-gamma contents. Thus, NE treatment significantly affected the H. contortus body and ESP expression, and changes in the ESPs influenced PBMC function. The results reveal a relationship between host hormones and parasites and provide new clues to explain some of the variation in individual responses to infection.

     

Effects of albendazole on Litomosoides chagasfilhoi (Nematoda: Filarioidea) females in vivo

Gerbils (Meriones unguiculatus) experimentally infected with Litomosoides chagasfilhoi were treated with a single oral dose of 40 or 80 mg of albendazole, respectively. Observation of the microfilaremia after the treatment showed that both single oral doses of albendazole decreased the microfilaremia in L. chagasfilhoi infection. The body wall was composed of a cuticle, a hypodermis, and a muscular layer, and treated nematodes showed no morphological alterations. The ultrastructural alterations produced by treatment with 40 mg of albendazole included a higher number of membrane invaginations in the basal labyrinth of the uterine epithelium and the presence of myelin figures in this region. Inside the uterus, most embryos and microfilariae were disintegrated. The treatment with 80 mg of albendazole did not produce alterations in the uterine wall, and the number of vesicles near the microfilariae sheath was smaller than that observed in the untreated and in the 40-mg treatment groups. However, all the microfilariae observed in the uterus were extensively damaged with cytoplasmic vacuolization and cellular degeneration. No alterations in the intestinal cells were observed after treatment with 40 or 80 mg of albendazole. The present study contributes to the knowledge of albendazole’s effects in filariids and demonstrates the potential embryotoxic and microfilaricidal consequences of this drug.     

Cyclohexadepsipeptides (CHDPs) with improved anthelmintical efficacy against the gastrointestinal nematode (Haemonchus contortus) in sheep

Besides 24-membered cyclooctadepsipeptides (CODPs) with the most prominent member of this class emodepside, the structurally related 18-membered cyclohexadepsipeptides (CHDPs) were of interest with regard to their efficacy against the nematode H. contortus in sheep.The CHDPs prepared by a simple total synthesis represent enniatin derivatives with strong in vivo activity against H. contortus in sheep. The correlation between the nature of the CHDP major conformers and their anthelmintic activities was studied in detail. All CHDPs with strong in vivo activity exists in deuterochloroform solution as conformers with restricted flexibility which was found by 2D-NMR spectroscopic analysis. This reduced flexibility of the major conformer can be exemplified by CHDPs containing e.g.: (i) an unsymmetrically folded conformation with no cis-amide bound, (ii) an internal hydrogen bond or (iii) one cis-amide bond, respectively.The strong in vivo anthelmintic activity against H. contortus in sheep indicates that the stereochemistry in 2-position of CHDPs is less important for their high inding affinity. It may be assumed that the identified inflexible region of the major conformers might mimic the active conformation of these CHDPs, which could be helpful for rational design of anthelmintics with less complicated structures.     

Ovicidal and larvicidal activity of crude extracts of Melia azedarach against Haemonchus contortus (Strongylida)

The rapid development of anthelmintic resistance, associated with the high cost of the available anthelmintic drugs, has limited the success of gastrointestinal nematodiosis control in sheep and goats and thus created interest in studying medicinal plants as an alternative source of anthelmintics. The aim of this study was carried out to evaluate the anthelmintic activity of the leaves and seed aqueous and hydro-alcoholic extracts of Melia azedarach L. (Meliaceae) were tested for in vitro ovicidal and larvicidal activity against Haemonchus contortus (Strongylida). Both extracts were evaluated at five concentrations: 12.5, 6.2, 3.12, 1.56, and 0.78 mg/ml. The leaves aqueous and hydro-alcoholic extracts inhibited 99.4% and 100% of the egg hatching and 100% of larval development at 12.5 mg/ml, respectively. In a similar way, the leaves hydro-alcoholic extract was the most active on egg inhibition (ED ₅₀ = 1.97 and ED ₉₀  = 5.05 mg/ml), leaves and seed aqueous and hydro-alcoholic extracts showed the best inhibition of larval development (ED ₅₀  = 3.01, 2.43, 3.17, 2.40, and ED ₉₀  = 10.53, 8.14, 11.94, and 8.19 mg/ml), respectively. These results suggest that utilization of M. azedarach extracts is useful in the control of H. contortus.     

Interactions of benzimidazoles (BZ) with tubulin from BZ-sensitive and BZ-resistant isolates of Haemonchus contortus

The binding of tritiated benzimidazoles (BZs)-albendazole, parbendazole, oxibendazole, mebendazole, oxfendazole and fenbendazole-to crude tubulin extracts from BZ-susceptible and -resistant Haemonchus contortus has been examined. For all BZs, the binding was substantially lower in the resistant isolate. The extent of this reduction was dependent on the structure of the BZ, with mebendazole demonstrating superior binding to the resistant isolate than the other BZs. Enrichment of the crude tubulin extract using polylysine-linked agarose demonstrated that for both isolates more than 85% of the observed binding was to protein eluted in the tubulin-containing fraction. Based on biochemical kinetics, the change in tubulin associated with resistance is reduced capacity in resistant tubulin to bind BZ with little or no reduction in the association constant of the BZ-tubulin complex. Comparative egg hatch assay demonstrated a similar structural specificity with the resistance factor of mebendazole observed to be lower than that of albendazole, parbendazole, oxibendazole and thiabendazole. The results of both studies support the hypothesis that BZ resistance is due to a change in tubulin and that this change is dependent on the structure of the BZ.     

Effect of concurrent and terminated infections of Haemonchus contortus on the development and reproductive capacity of Nematodirus battus

Both concurrent and chemically terminated infections of Haemonchus contortus were found to lower the take and egg producing capacity and inhibit the development of subsequent infections of Nematodirus battus.     

Beta-tubulin and benzimidazole resistance in the sheep nematode Haemonchus contortus

We have compared benzimidazole (BZ) susceptible (s) and resistant (R) strains of Haemonchus contortus from sheep by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), Western blotting and ELISA techniques. The S strain bound more drug per mg protein than the R strain. BZ binding could be resolved into high-affinity and low-affinity binding. Low-affinity binding in parasite preparations devoid of tubulin was observed, but high-affinity binding occurred only in preparations containing tubulin. Resistance was associated with a decrease in the high affinity component. The S and R strains were shown by ELISA to contain similar total amounts of tubulin. By 2-D PAGE, the beta-tubulin isoform pattern of the S strain was different from that of the R strain, but the alpha-tubulin isoform patterns of the 2 strains were similar. BZ resistance was associated with a decrease in high-affinity BZ binding to tubulin and an alteration in beta-tubulin isoform pattern.     

Microsatellite diversity of isolates of the parasitic nematode Haemonchus contortus

The alarming development of anthelmintic resistance in important gastrointestinal nematode parasites of man and live-stock is caused by selection for specific genotypes. In order to provide genetic tools to study the nematode populations and the consequences of anthelmintic treatment, we isolated and sequenced 59 microsatellites of the sheep and goat parasite Haemonchus contortus. These microsatellites consist typically of 2-10 tandems CA/GT repeats that are interrupted by sequences of 1-10 bp. A predominant cause of the imperfect structure of the microsatellites appeared mutations of G/C bp in the tandem repeat. About 44% of the microsatellites were associated with the HcREP1 direct repeat, and it was demonstrated that a generic HcREP1 primer could be used to amplify HcREP1-associated microsatellites. Thirty microsatellites could be typed by polymerase chain reaction (PCR) of which 27 were polymorphic. A number of these markers were used to detect genetic contamination of an experimental inbred population. The microsatellites may also contribute to the genetic mapping of drug resistance genes.     

Cytological studies on the absorptive surfaces of cestodes

Summary Autoradiographic studies of amino acid incorporation by Hymenolepis diminuta indicate high levels of protein synthetic activity in the subcuticular integumentary cells. Two hours after receiving a 5 minute pulse of tritiated proline, a significant percentage of the radioactivity initially incorporated into the subcuticular cytoplasm appears in the cuticular matrix. The results suggest that structural and non-mitochondrial enzymic proteins of the cuticle are synthesized in the subcuticular cells then secreted into the cuticular matrix. This interpretation is consistent with the cytology of the cestode integument revealed by electron microscopy.

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Zusammenfassung Autoradiographische Studien mit Aminosäuren, die Hymenolepis diminuta inkorporiert wurden, zeigen den hohen Grad der Protein-Syntheseaktivität in den Zellen des subcuticulären Integuments an. Zwei Stunden nach Applikation von tritiummarkiertem Prolin tritt in der cuticularen Matrix ein bedeutender Anteil der radioaktiven Substanz in Erscheinung. Die Resultate lassen vermuten, daß den subcuticulären Zellen strukturelle und enzymatische Proteine der Cuticula synthetisiert werden und dann in die cuticulare Matrix gelangen. Diese Interpretation steht in Übereinstimmung mit den cytologischen Befunden am Cestodenintegument, wie sie durch die Elktronenmikroskopie gewonnen wurden.

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This study was supported in part by grants from the National Institutes of Health, U.S. Public Health Service (5 TI AI 106 and AI 01384) and the American Cancer Society (IN-24-G). Portions of this investigation were conducted under the tenure of a predoctoral fellowship (1-Fl-GM-23, 449) from the U.S. Public Health Service.     

Protective immunity to a blood-feeding nematode (Haemonchus contortus) induced by parasite gut antigens.

To determine the ability of gut antigens to induce a protective immune response against blood-feeding nematodes, isolated gut antigens were used to immunize goats against Haemonchus contortus. Immunization-induced antibody responses recognized parasite gut antigens which were associated predominantly with the microvillous membrane region of the parasite gut. Antibody from immune serum also recognized seven predominant gut proteins on a Western blot (immunoblot). Several of these proteins appeared to be integral membrane proteins on the basis of their solubility in the detergent Triton X-114, indicating that the presentation protocol stimulated an antibody response to microvillous membrane antigens. Three different age groups of goats ranging from less than 6 months to greater than 1 year were immunized for challenge experiments. After infection with 10(4) larvae, an 87 to 95% reduction in fecal egg counts for all age groups of goats was achieved in the immunized compared with the control group. The reduction of worms in immunized goats ranged from 65% (kids) to 89% (yearlings) compared with controls. These results indicate that gut antigens can induce significant protection against blood-feeding nematodes. Antibody to H. contortus gut antigens also cross-reacted with microvilli of other blood-feeding nematodes including Ostertagia ostertagi and small strongyles of horses, which indicates that epitopes associated with the gut are phylogenetically conserved.     

Histochemical studies on the body wall of nematodes: Haemonchus contortus (Rud., 1803) and Xiphinema insigne Loos, 1949

Summary Histochemical studies on the body wall of Haemonchus contortus (Rud.) and Xiphinema insigne Loos have been made. In H. contortus, the cuticle is mainly proteinous in nature. The lipids and PAS-positive materials are only present in cortical layers. In addition, haemoglobin and acid phosphatase are also present. The hypodermis shows the presence of glycogen, lipids, RNA, acid and alkaline phosphatases. The oval dense body is composed of keratinous and collagenous proteins associated with acid mucopolysaccharides. Muscles carry a greater concentration of glycogen granules and phospholipids. In X. insigne, the cuticle is rich in sudanophilic lipids. The cuticle also consists of weakly acidic mucopolysaccharides. Hypodermis and muscles contain lipids and glycogen. In addition, hypodermis also consists of acidic mucopolysaccharides. The functional significance of these components has been fully discussed.     

Identification and preliminary characterization of cuticular surface proteins of Haemonchus contortus

Cuticular surface antigens of the XL3 and L4 stages of Haemonchus contortus have been studied by surface labeling and immunological techniques. Live worms were labeled with 125I and extracted with sodium dodecyl sulfate (SDS) followed by SDS + 2-mercaptoethanol. The SDS-soluble surface proteins of XL3s and L4s were found to consist of relatively few major species. The pattern of labeled polypeptides was distinctive for each developmental stage. These proteins are refractory to digestion by bacterial collagenase. Several of the proteins are glycosylated. Further extraction of labeled worms with SDS + 2-mercaptoethanol solubilized additional labeled proteins that appeared to be primarily collagens. Rabbit antisera prepared against native XL3 and L4-cuticles reacted strongly with the surfaces of live worms in immunofluorescence assays. In contrast, antisera prepared against SDS-extracted cuticles reacted weakly or not at all with live worms in similar experiments. Rabbit antisera prepared against adult cuticles failed to react with live XL3s or L4s. These studies suggest that the major surface antigens of XL3s and L4s are solubilized by SDS and that there are different antigens present on the cuticular surfaces of XL3s, L4s and adults. Stage-specificity in cuticular surface proteins may contribute to the successful parasitic lifestyle of this nematode.     

Isolation and characterization of class II myosin genes from Haemonchus contortus

In this study, cDNAs encoding myosin from the parasitic nematode Haemonchus contortus were isolated and characterized. Several exhibited a considerable degree of sequence variation at the nucleotide and limited divergence at the amino acid levels within the various functional domains. The results suggest that the cDNAs isolated represented a single myosin heavy chain, which, by comparison with a number of other myosins, is inferred to represent a homologue of a muscle myosin (CeMHCA) of the free-living nematode Caenorhabditis elegans. The findings could have implications for investigating cytoskeletal dynamics and/or signalling pathways.     

Effects of excretory/secretory products of Haemonchus contortus on cell vacuolation

Excretory/secretory (ES) products of the gastric nematode, Haemonchus contortus, have been implicated in the inhibition of gastric acid secretion which follows infection. Parietal cell vacuolation has been observed in abomasal sections from parasitised sheep, and ES prepared in vitro has been reported to cause vacuolation and to increase neutral red (NR) uptake in epithelial cell cultures. We have used the latter approach to examine, at the cellular level, the effects of ES prepared from L3 and adult nematodes. Both NR uptake and cellular vacuolation were increased following exposure to larval or adult ES products. ES preparations from adult worms induced more extensive vacuolation then those from L3 worms and, as with VacA treatment, adherent cells remained viable despite high levels of vacuolation. Whereas VacA-induced vacuolation resulted in NR uptake predominantly localised in vacuoles, this appeared not to be the case with ES-induced vacuolation, suggesting that different mechanisms might be involved. Both ES and VacA exposure was associated with an increased rate of cell detachment.     

Effects of Myracrodruon urundeuva extracts on egg hatching and larval exsheathment of Haemonchus contortus

The anthelmintic resistance has limited the control of gastrointestinal nematodes of small ruminants and thus has awakened interest in the study of tanniferous plants as a source of anthelmintics. These experiments were carried out to evaluate the in vitro efficacy of Myracrodruon urundeuva leaf extract (LE) and stem extract (SE) against Haemonchus contortus. An inhibitor of tannins, polyvinyl polypyrrolidone (PVPP), was used to verify if these metabolites are involved in the anthelmintic activity of the extracts. To evaluate the ovicidal effect, H. contortus eggs were incubated with the extracts (0.31 to 5 mg/mL) for 48 h. In the larval artificial exsheathment assay, third-stage larvae of this nematode were incubated with extracts (0.31 mg/mL) for 3 h and then were exposed to a sodium hypochlorite solution. The exsheathment process was evaluated for 60 min. The results were subjected to the Kruskal–Wallis test (P < 0.05). The extracts showed dose-dependent ovicidal effects, although the LE was more effective, inhibiting egg hatching by 97.73% at 1.25 mg/mL, while the SE inhibited hatching by 83.56% at 5 mg/mL. Contact with the extracts blocked the larval exsheathment (P < 0.05). The addition of PVPP confirmed the role of tannins, as there was a substantial reduction in egg hatching and larval exsheathment percentage. These results suggest that M. urundeuva can be used to control gastrointestinal nematodes of small ruminants and that the anthelmintic activity of this plant is probably related to tannins; however, in vivo studies should be conducted.     

Characterization of local antibody responses to the gastrointestinal parasite Haemonchus contortus.

The ability to identify antigens associated with an infection has generally relied on the use of serum antibodies produced by infected or previously exposed individuals. A major drawback with the use of serum is that it does not necessarily reflect the local antibody response at mucosal tissue sites. This study describes an approach that allows the use of antibodies generated close to the infection site to detect the transient expression of stage-specific antigens during infection with the gastrointestinal parasite Haemonchus contortus. This was achieved by infecting immune sheep with H. contortus larvae and removing the abomasal lymph nodes draining the infection site shortly after the challenge infection. Antibody-secreting cell (ASC) probes were generated from these lymph nodes after short-term in vitro culture of cell suspensions, which allowed the accumulation of antibodies secreted by in vivo-induced ASC into the culture supernatant. Lymph node culture supernatants (= ASC probes) from immune sheep challenged 5 days previously were used to probe Western blots of third and fourth stage larval preparations, and revealed distinct reactivity to larval antigens. No antibody reactivity to larval antigen preparations was detected in sheep that were not challenged. The number of antigens identified using ASC probes was significantly restricted compared to either pre- or post-challenge sera. In contrast to the variability of the serum response, the specificity of ASC probes was highly repeatable between different sheep. ASC probes were also used to purify a H. contortus larval antigen by affinity chromatography, which allowed limited biochemical studies to be undertaken. The antigen(s) recognized by the ASC probes were shown to be expressed on the surface of the larvae. These studies illustrate the use of a novel means of studying the local antibody response close to a mucosal infection site in order to identify and isolate stage-specific antigens expressed during infection.     

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.     

A comparative study on clinical pathology changes in experimentally infected sheep with active and arrested larvae of Haemonchus contortus

Hypobiosis is the larval arrest of parasites and is an adaptation to host and environmental conditions. This study was conducted to compare clinicopathological changes in sheep experimentally infected with fresh and arrested larvae of Haemonchus contortus. Twenty-eight apparently healthy 6-month-old Shal lambs, whose stool samples were eggs per gram (EPG) negative, were divided into four groups: A, B, C and D. Groups A and D received fresh larvae and placebo, respectively. Treatment groups (B and C) were infected by arrested larvae obtained under different conditions such as humidity (B, 70%; C, 40–50%), temperature (B, 8–10°C; C, 35–37°C) and light intensity (B, low; C, high). Clinical signs were monitored until day?60, haematological examination [hematocrit (Hct), haemoglobin (Hb), red blood cells (RBC), mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), white blood cells (WBC) and differential cell count] and biochemical examination (Ca, Mg, P, total Pro, Alb, Globulins and Alb/Glob ratio) were performed on the first day and 2?months after infection with the larvae. In addition to the faecal egg count, the number of larvae and adult worms were determined in abomasal contents. The mean number of adult worms and faecal egg count in group A were significantly higher than in other groups (p?<?0.01). The proportion of arrested larvae and percentage of hypobiosis in group B differed significantly from other groups (p?<?0.05). The body weights of group A, B and C were significantly different compared with group D (p?<?0.01). No difference in weight gain was observed between the treatment groups B and C. Considerable reduction in RBC, Hb and PCV were observed in the different treatment groups, but these reductions were highly significant in group A (p?<?0.05) while MCV, MCH and MCHC did not show any change. The comparison of total WBC and differential cell count between groups indicated the presence of eosinophilia in group A (p?<?0.0005). Serum protein, albumin and calcium concentration decreased only in group A (p?<?0.01).     

Cuticle collagen genes of Haemonchus contortus and Caenorhabditis elegans are highly conserved

Several genes and partial cDNAs encoding cuticle collagens have been isolated from the sheep parasitic nematode Haemonchus contortus. DNA sequencing and Southern blot hybridization studies reveal that H. contortus collagens comprise a large family of related, but non-identical genes. The genes appear to be dispersed throughout the genome. The predominant size of collagen mRNA in molting worms was found to be between 1.0 and 1.2 kb. The one complete gene that was sequenced contains two short introns and encodes a protein of about 300 amino acids. The predicted protein sequence contain several (Gly-X-Y)n triple helix-coding domains that are interrupted by short stretches of non-helix-coding amino acids. The size of the predicted protein and the organization of the triple-helix coding domains are similar to that of Caenorhabditis elegans collagens. All the H. contortus genes studied show a striking homology to the C. elegans collagen gene subfamily represented by col-1. In particular, the amino acid sequence of the carboxy-terminal non-(Gly-X-Y)n region and the positions of cysteine residues flanking the (Gly-X-Y)n domains were found to be highly conserved in the collagens of these two nematodes.