Haemonchus contortus: a simple procedure for purifying surface proteins from third- and fourth-stage larvae.

Surface proteins were solubilized from exsheathed third (XL3)- and fourth (L4)-stage larvae of Haemonchus contortus by a one-step extraction procedure involving brief heat treatment of the worms in the presence of buffer and 100 mM sodium chloride. Surface proteins also could be preferentially extracted from XL3s, but not from L4s, by heating the worms briefly in 1% sodium dodecyl sulfate. The major proteins extracted by these procedures were similar in molecular weight to those detected by surface-labeling live worms with 125Iodine. Both extraction procedures solubilized a single, major protein with an apparent molecular weight of 68-97 kDa from XL3s. In contrast, extraction of L4s with 100 mM sodium chloride yielded four major proteins with relative molecular weights of 27, 29, 78, and 200 kDa. Antibodies raised in rabbits to surface proteins prepared by the sodium chloride procedure reacted with the surfaces of live worms in indirect immunofluorescence assays. The anti-XL3 surface protein serum was stage specific in immunofluorescence experiments using live worms and in immunoprecipitation experiments using 125Iodine-labeled XL3 and L4 surface proteins. The overall amino acid composition of the surface proteins is hydrophilic. Twenty-six percent of the amino acid residues of the XL3 surface proteins, which consist predominantly of the 68-97 kDa species, are glutamate or glutamine.

Biochemical characterization of cuticle polypeptides from the infective larvae of Haemonchus contortus.

1. Ecdysis of infective Haemonchus contortus larvae is effected by the enzymatic degradation of a specialized region of the second molt cuticle containing a biochemically unique polypeptide (mol. wt = 160,000). 2. The 160,000 mol. wt polypeptide and related polypeptides are synthesized at approximately 6 days of larval development. Antigenically similar polypeptides occur in other ruminant trichostrongyles. 3. Cuticle polypeptides digested during ecdysis differ from second molt cuticle collagens in amino acid composition and collagenase sensitivity. However, some antigenic homology between the 160,000 mol. wt polypeptide and cuticle collagens suggests structurally similar regions.

The cuticular proteins from free-living and parasitic stages of Haemonchus contortus--I. Isolation and partial characterization.

1. Cuticles were isolated from the adult males, adult females, the second molt (2M) sheath from the infective larvae (L3(2M)), and the parasitic third stage (L3) of the sheep parasite Haemonchus contortus by a combination of mechanical disruption and detergent treatment. 2. The 2ME soluble cuticular proteins from adult males contained 4 or 5 major protein bands with molecular weights ranging from 100 to 56 kD with the most prominent band at 56 kD. The cuticular proteins from adult females were similar to the male. 3. Cuticular proteins from the larval stages, 2M cuticle, and L3 cuticle, differed from the adults and from each other. The most prominent protein bands were observed with molecular weights on 78 and 39 kD for the L3 cuticle and 100, 91 and 46 kD for the 2M cuticle. The 2ME soluble cuticular proteins from all developmental stages were at least partially digested by bacterial collagenase. 4. The amino acid composition of cuticular proteins was similar for the L3 and 2M, but adults had lesser amounts of glycine and greater amounts of basic amino acids than the larval stages. The amount of the isolated cuticle solubilized by the 2ME treatment was greatest in adults (80%) compared to the L3 (64%) and the 2M (22%). 5. These results support a hypothesis that there are quantitative and qualitative stage specific differences in the cuticular proteins of H. contortus.

Cuticular carbohydrates of three nematode species and chemoreception by Trichostrongylus colubriformis.

The cuticle of Haemonchus contortus, Nippostrongylus brasiliensis and Trichostrongylus colubriformis contained N-acetylgalactosamine and N-acetylglucosamine, based on the binding of fluorescein-labelled lectins. Binding of the lectins varied between the sexes and body regions of the nematodes. Treatment of male T. colubriformis with the lectin Lens culinaris agglutinin (LcA) reduced the feeding by helminths that was stimulated by histamine and the male's response to their female's pheromone, based on in vitro assays. Mannose residues may be involved in the helminth's chemoreceptors for feeding and sexual attraction, based on the specific binding of LcA.

Manifestations of resistance to ovine ostertagiasis associated with immunological responses in the gastric lymph.

Groups of previously infected and worm-free sheep were serially killed up to 10 days after challenge with 50 000 Ostertagia circumcincta larvae. Two similar groups of sheep were killed 10 days after challenge with 1000 larvae. The previously infected sheep were resistant to the smaller challenge dose in that fewer, stunted worms were recovered from them than from controls. However, this resistance was not as marked as that observed in the previously infected sheep which received the large challenge, because proportionally fewer worms were recovered after the 50000 dose and the great majority of these were arrested at the early fourth stage. The gastric lymph ducts of 6 previously infected sheep were cannulated successfully and a marked local immune response was detected in 3 sheep which were challenged with 50 000 larvae. No response was detected in 3 cannulated sheep challenged with 1000 larvae. In the lymph of the 50 000 dose group, a temporary increase in pepsinogen activity suggested that a hypersensitivity reaction related to the presence of large numbers of mucosal mast cells began between 24 and 48 h after challenge. This was followed by marked increases in the cellular and IgA content of lymph, which reached peaks on days 3 and 6, respectively. It is suggested that the response detected in the gastric lymph reflected aspects of a local immune reaction in the abomasal mucosa and that this reaction accounted for the enhanced degree of resistance to the larger challenge dose.

Partial biochemical and serological characterization of exsheathing fluid and somatic extracts of infective larvae and adult females of Haemonchus contortus.

Exsheathing fluid from infective larvae of Haemonchus contortus may contain antigens which induce protective immunity upon infection in sheep. In this study, a rapid method of preparing exsheathing fluid was used to determine its biological activity, purity and antigenic complexity. Protein in exsheathing fluid ranged from 21-59.5 micrograms/mL and no carbohydrate was detected. Gel filtration showed a single broad peak which retained biological activity and polyacrylamide gel electrophoresis demonstrated a single band staining with Coomassie blue. Biological activity of exsheathing fluid was present in extracts of infective larvae but not in extracts of exsheathed larvae or in extracts of adult female H. contortus. Rabbit antiserum against exsheathing fluid proteins cross-reacted with infective larvae extracts but not with adult female worm extracts. Immunoelectrophoresis of exsheathing fluid proteins against this antiserum showed a single polydisperse precipitin line. Thus, exsheathing fluid produced by the rapid method is a pure preparation of a biologically active and antigenically simple material suitable for further studies of protective immunity in sheep to H. contortus.

Inhibition of gastric hydrochloric acid secretions in the rat given Ostertagia ostertagi (a gastric parasite of cattle) extract.

The immersion of freshly collected Ostertagia ostertagi adults into saline solution (NaCl, 0.9%; pH 1.0) resulted in 100% mortality of worms within a 10-minute period. At pH 7.0, mortality was 58% after 270 minutes. At pH 2.2, which approximates the usual abomasal pH, 100% mortality resulted within 60 minutes. In the stomach of rats injected with an extract of O ostertagi (from the abomasum of cattle), there were decreased total secretion of hydrogen ions and volume of secretion (P less than 0.05) and increased mean pH of gastric secretion (P less than 0.05). These effects were similar to those found in stomach of rats treated with cimetidine. Results indicated that the pH of the abomasum was detrimental to parasitic survival and that hypochlorhydria found during ostertagiosis may be mediated partially by a chemical released from the parasite.

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

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-postive 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.

Observations on crystals found in the intestine of Nematodirus battus during the development of immunity to this nematode in lambs.

Lambs which were given 60000 infective larvae of Nematodirus battus progressively threw off adult nematodes from day 20 onwards. Large, hexagonal crystals appeared in the intestine of the adult nematode at about this time and caused blockage of the intestinal-rectal or -cloacal junction. Lambs which were given 1000-2000 infective larvae did not throw off the infection, and adult nematodes from lambs which had been infected for up to 74 days did not contain these crystals. Chemical, histochemical and x-ray microanalysis tests on the crystals indicated that they are lipoprotein in composition. The crystals arise within the lumen of the nematode and appear to be associated with the development of immunity to this nematode in lambs.