Preliminary characterization and interaction of tubulin from Trichinella spiralis larvae with benzimidazole derivatives.

Tubulin was estimated to account for 0.3% of the total soluble protein in Trichinella spiralis cytosolic fractions. Tubulin from T. spiralis was partially purified by precipitation with either taxol or vinblastine sulphate. Immunoblotting with alpha- and beta-tubulin monoclonal antibodies revealed the presence of tubulin in T. spiralis partially purified preparations. Electrophoretic mobility of T. spiralis tubulin in sodium dodecyl sulphate-polyacrylamide gels was very similar to that shown by pig brain tubulin. Further studies with colchicine binding assays indicated that T. spiralis tubulin has binding features similar to that of tubulin from other nematodes: colchicine association constant = 8.1 x 10(-4) M and competitive inhibition of colchicine binding by podophyllotoxin, with an inhibition constant of 1.3 x 10(-6) M. Finally, inhibition of colchicine binding by several benzimidazoles (mebendazole, fenbendazole, oxibendazole and albendazole) was investigated. All the benzimidazoles inhibited colchicine binding in a competitive manner, with inhibition constant values ranging from 1.4 x 10(-7) M (mebendazole) to 3.9 x 10(-6) M (fenbendazole).

Anatomical location of phosphorylcholine and other antigens on encysted Trichinella using immunohistochemistry followed by Wheatley's trichrome stain.

This work investigated the location on the parasite of Trichinella antigens recognized by the mouse immune system and the question as to which of them bear the epitope phosphorylcholine (PC). Wheatley's trichrome stain (initially developed for faecal smears) proved to be excellent for visualization of Trichinella structures, enabling four types of stichocyte to be distinguished. By applying this stain on infected muscle sections after immunocytochemistry using (a) anti-PC BH8 monoclonal antibodies, (b) serum from mice that had been infected twice in the presence of 0.05% thiabendazole (to prevent reproduction by adult females) and then bled on day 7 post-reinfection, (c) serum from infected mice that were bled on day 14 postinfection, or (d) serum from infected mice that were bled on day 42 postinfection, we found (1) that PC is an abundant structural epitope on the hypodermis/muscle, genital primordium and intestinal tract but is absent from the cuticle and stichosome; (2) that the principle secretory cells of adult worms are delta- and beta-stichocytes, whereas those of migrating and encysted L1 larvae are alpha-stichocytes; and (3) that Trichinella antigens recognized in the encysted phase of the parasite's life cycle are present in parasitized myofibres in the sarcoplasmic matrix and in the nucleoplasm of hypertrophic nuclei. The significance of these findings is discussed.

An analysis of Trichinella pseudospiralis excretions and study of their effect on striated muscles of mouse.

The causes of changes observed after infection of muscles with Trichinella pseudospiralis larvae were studied. The changes are not only regressive, but also proliferative (activation and mitosis of satellite cells and formation of new myotubes). By means of high-performance liquid chromatography (HPLC) and an original method it was found that T. pseudospiralis larvae excrete into their environment among others a great amount of n-butylamine. A direct application of n-butylamine into the gluteal muscle of mouse supported the assumption that this amine is one of the factors causing the changes in muscles induced by T. pseudospiralis infection. The application of n-butylamine into the muscle resulted in regressive and regenerative changes similar to that observed in case of T. pseudospiralis infection.

[Intrastrain heterogeneity of Trichinella spiralis: a sedimentation analysis]. / Vnutrishtammovaia geterogennost' Trichinella spiralis: sedimentatsionnyi analiz.

Intrastrain heterogeneity of muscular larvae of trichinellids has been revealed in experiments of free sedimentation and isopyknic division in the density gradient of saccharose, the presence of which is confirmed by the study of morphological characters and infection activity. The presence of intrastrain heterogeneity indicates the necessity of introduction of standard technique for studies of biological characters of different strains of trichinellids.

Characterization of the accessory layer of the cuticle of muscle larvae of Trichinella spiralis.

The accessory layer of the cuticle of infective larvae of Trichinella spiralis has been studied with electron microscopy using cytochemical techniques and chemical extractions. The accessory layer lacks negative charges and carbohydrates demonstrable in vivo. Staining with ruthenium red and tannic acid is interpreted as being consistent with their reactions with phospholipids. Freeze fractures demonstrate an external layer of granules that can be partially released by means of detergents (CTAB and SDS). The granules are considered to be proteins. Their removal makes the worms acid sensitive and prevents them from infecting mice. Extraction of whole worms with ethanol, acetone and methanol (via reaction with 2,2-DMP), or chloroform and methanol destroys an internal layer of filaments. Thin-layer chromatography of chloroform/methanol extracts showed principally ethanolamine phospholipids from the surface of the worms. A model is presented for the molecular organization of the accessory layer. Ethanolamine phospholipids are suggested to occur as tubular micelles. Proteins may attach to these by lipophilic moieties and perhaps by a cryptic sugar group (demonstrated by others) that may penetrate into the hydrophilic core of the lipid micelles.

Carbohydrate reserves and infectivity in Trichinella spiralis isolated from carrion.

Trichinous mice were killed and their carcasses maintained at room temperature or subzero temperatures for varying lengths of time. Some worm parameters were measured after direct isolation from carcasses while others were measured following passage through a second round of hosts. Glycogen and trehalose levels in infective 1st-stage larvae (L1) isolated directly from carcasses maintained at room temperature were significantly less than controls (day 0) after day 4 post-kill (p.k.). By day 21 p.k. among L1 isolated directly from mouse carcasses those observed coiled or moving had decreased by around 20% compared to day 0 p.k. The percentage of L1 isolated from carcasses on several days p.k., used to infect mice and recovered as pre-adult worms declined significantly after they had remained in carcasses for 14 days. Only 2.6% of muscle larvae isolated from carcasses on day 21 p.k. and used to infect mice were recoverable as pre-adults. Pre-adult worms raised in mice infected with larvae from day 7 carcasses had about 50% less glycogen than worms raised in mice infected with larvae isolated from fresh carcasses (day 0 p.k.). The fecundity in vitro of adult worms isolated from mice on day 7 following infection with infective L1 larvae maintained in carcasses held at room temperature for 0-16 days declined only when adult worms developed from larvae recovered from carcasses at 3 days following host death. Following 24 h at temperatures below freezing, infectivity of L1 larvae isolated from frozen carcasses was reduced by 97%. Carbohydrate levels remained high in larvae from carcasses frozen for up to 4 days.

Comparative study on polypeptide patterns of larvae of Trichinella isolates by two-dimensional electrophoresis.

The two-dimensional patterns (isoelectrofocusing-IEF/polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate-SDS) of S3 fractions of muscle larvae of four Trichinella isolates were compared. The comparative study concerned six groups of polypeptides. It was observed that the Garkavi isolate of Trichinella pseudospiralis was clearly different from the other isolates, and it showed the simplest IEF/SDS polypeptide pattern. The C-76 isolate of T. nelsoni had only four of the six groups, distinguishing it from the GM-1 isolate of T. spiralis and the Boev isolate of T. nativa that showed all the indicated groups.

Trichinella spiralis: behavior, structure, and biochemistry of larvae following exposure to components of the host enteric environment.

Four layers are present on the surface of infective larvae of Trichinella spiralis isolated from host muscle in pepsin-HCl. Trypsin treatment of pepsin-HCl isolated worms caused partial degradation and removal of large patches of the two outer surface layers. Following exposure to bile, only traces of the outer layers remained on the worms surface. These changes in the worm surface were accompanied by a shift from Type I behavior, typical of pepsin-HCl isolated larvae, to Type II behavior, (snakelike) following exposure to either trypsin or bile. Worm behavior was also temperature dependent. Type I behavior was typical of worms maintained at room temperature regardless of treatment, while Type II behavior displayed by worms held at 37 C was treatment dependent. The absorption of in vitro glucose or beta-methyl-D-glucoside was lowest in pepsin-HCl isolated first stage infective larvae, significantly higher in trypsin treated worms and greatest in worms following exposure to bile. Sugar uptake by worms isolated from the host small intestine after 1 hr of enteral infection was similar to that seen in worms isolated from host muscle in pepsin-HCl. Sugar uptake in vitro in worms 2 hr following enteral infection was similar to worms following exposure to bile. The highest levels of sugar absorption in vitro occurred in worms which had resided in the small intestine for 3 hr. The lowest rates of incorporation of label into worm tissues was seen in 1 hr enteral and pepsin-HCl isolated worms. Infective larvae treated with trypsin or bile incorporated significantly greater amounts of label than the two former groups. The highest levels of incorporation of label into worm tissues was seen in 3 hr enteral worms. These findings support the view that trypsin, bile, and temperature serve as environmental cues which lead to alteration of the parasite's behavioral and nutritional status.

Biophysical properties of the surface lipid of parasitic nematodes.

The biophysical properties of the surface lipid layer (the epicuticle) of living parasitic nematodes (Trichinella spiralis and Toxocara canis) were examined using fluorescent lipid analogues. A variety of such probes were screened, and only 5-N-(octadecanoyl)-aminofluorescein was found to insert into the outer lipid layer. Fluorescence quenching experiments showed that this probe was confined to the surface, and the rate of its lateral diffusion was then measured by Fluorescence Recovery After Photobleaching. This showed that the probe was not free to diffuse within the plane of the epicuticle. This structure is, therefore, extraordinary in its selectivity to lipid probes, and in the restricted lateral mobility of inserted lipid components.

Comparative studies on soluble protein profiles and isozyme patterns of seven Trichinella isolates.

Soluble protein profiles and isozyme patterns of eight enzymes were compared for extracts of muscle stage larvae of the seven Trichinella isolates, using isoelectric focusing in polyacrylamide gel. Soluble protein profiles and isozyme patterns of four enzymes: malic enzyme, glucosephosphate isomerase, phosphoglucomutase, superoxide dismutase of them were clearly divided into four types. T. pseudospiralis from a racoon and the Polar strain from a polar bear formed type 1 and type 2. The Iwasaki strain from a Japanese black bear and the Yamagata strain from a racoon dog, both from Japan, were type 3. Type 4 consisted of three remaining strains, the Polish strain from a wild pig, the USA strain from a pig and the Thai strain from a human case, which have similar infectivities to pigs. The Thai strain varied a bit electrophoretically from other members of type 4. Zymograms of adenylate kinase and malate dehydrogenase were similar in types 2 and 3. The 6-phosphogluconate dehydrogenase zymogram of type 3, similar to that of type 4, was different from that of type 2. It is assumed from the data that type 3 (Japanese strain) was genetically intermediate to types 2 and 4. T. pseudospiralis and the Polar strain had a common main isozyme of 6-phosphogluconate dehydrogenase. The zymogram of lactate dehydrogenase was common except for T. pseudospiralis.

A comparison of the surface and secretions of Trichinella pseudospiralis and T. spiralis.

Intact, viable adults, infective and newborn larvae of Trichinella pseudospiralis were surface labelled with 125I by the chloramine T method and labelled proteins were compared with those obtained from equivalent stages of T. spiralis. Electron-microscope autoradiography determined that labelled proteins were restricted to the cuticle for all stages of both isolates. Comparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE), using thin gradient gel slabs, of proteins obtained from each stage, demonstrated that the profile of surface-labelled proteins of T. pseudospiralis were restricted in number, stage specific, and similar to equivalent proteins of T. spiralis both in size and in their organization into aggregates. The stage-specific profiles of surface-labelled proteins derived from newborn larvae were indistinguishable, but differences were noted between adults and infective larvae of the two isolates. These differences in protein structure were confirmed by two dimensional mapping of tryptic peptides. Stage-specific profiles were also obtained when [35S]methionine biosynthetically labelled secretions of the 3 stages of T. pseudospiralis were compared by SDS-PAGE. Comparison of the profiles obtained with secretions for respective stages of T. spiralis again failed to distinguish newborn larvae, but adults and infective larvae of T. spiralis and T. pseudospiralis displayed a mixture of common and species-specific proteins. These findings are discussed in relation to the different pathology associated with infection with two isolates.

Histochemical differentiation of four species of the genus Trichinella from days 10-40 of their development in muscles of experimentally infected mice.

Differences in an infection of the muscle caused by larval T. pseudospiralis (from days 10-40 p.i.) and that caused by capsule-forming Trichinella larvae were disclosed with histochemical techniques. These were: An intense reaction of the modified sarcoplasm for neutral mucosubstances, and intense staining of spherical centres of re-differentiation in the sarcoplasm for tyrosine, activity of the acid phosphatase, a negative reaction for SS groups of proteins at the site of location of the parasite owing to the absence of a collagen-like substance deposited by capsule-forming Trichinella species. Alkaline phosphatase activity weak in a sarcoplasm infected with larval T. pseudospiralis, but intense in capsule-forming species suggesting a different, larval metabolism. The capsule of T. nativa which is produced later than that of T. spiralis and T.nelsoni, differs from these also histochemically in that it contains neutral polysaccharides on the surface of the outer capsule layer which stains for non-sulphonated, acid mucosubstances (hyaluronic acid). Inspection with the electron microscope disclosed a vacuolate substance containing glycoprotein, phospholipids, and displaying acid phosphatase activity. The substance was present in the vicinity of larvae of T. nativa occupying the space between the cuticle and the sarcoplasm. A slight, morphological difference between T. nelsoni and T. spiralis was observed in the more elongate shape of the capsule at days 30 and 40 p.o. Histochemical differences were in an oxidative reaction with aldehyde fuchsin (PAA AF, KMnO4 A F) showing an increased hypertrophy of the connective tissue surrounding muscle fibres infected with T. nelsoni.

Strongyloides ratti and Trichinella spiralis: net charge of epicuticle.

The intact epicuticles of Strongyloides ratti stage-3 larvae and Trichinella spiralis stage-1 larvae were found to have a surface net negative charge. Ultrastructural studies on S. ratti using cationized ferritin and ruthenium red showed the negative charge to be dense and uniformly distributed over the epicuticular surface. Staining with acetic acid-ferric oxide hydrosol occurred at pH 1.65 and suggests that amino acid carboxyl groups were not responsible for the negative charge property. Alcian blue staining occurred at pH 0.5 and at a critical electrolyte concentration (CEC) of 0.9 M MgCl2, a property similar to that of highly sulfated mucopolysaccharides such as the proteoglycan keratan sulfate. In contrast, T. spiralis larvae failed to stain with alcian blue below pH 5.0 or at a CEC of 0.1 M, suggesting its negative charge is associated with dissociated amino acid carboxyl groups. Attempts to remove the negative charge-bearing components in the epicuticle of S. ratti by detergents, organic solvents, denaturing agents, proteases, uronidases, neuraminidases, and lipases were unsuccessful. The presence of elastin in the S. ratti larval outer cortical layer was indicated by its vulnerability to elastase and its reaction to aldehyde fuchsin-alcian blue stain. These results show that the epicuticle of S. ratti is not a typical cell membrane, although it appears to have ultrastructural similarities. It is suggested that the association of highly sulfated mucopolysaccharides with the epicuticular surface of free-living nematodes such as S. ratti L3 may reflect a greater need to protect against surface desiccation. It is also postulated that the highly negatively charged surface may have anticomplementary and anticoagulation effects.

Comparative electron microscopic and cytochemical studies on the cuticle and the hypodermis of Trichinella nativa (Britov et Boev, 1972) and T. pseudospiralis (Garkavi, 1972).

Using cytochemical methods, we disclosed both in the cuticle and the hypodermis of two-day-old females of T. nativa and T. pseudospiralis, in the area of the oesophagus, these substances: Cystine-containing proteins of the SS group, carbohydrates, acidic mucopolysaccharides (AMS), acid phosphatase (ACP) and a nonspecific esterase (NE). A regularly transverse striation (striae) in the cuticle reacts strongly with the method for cystine-containing proteins, weakly for carbohydrates, and negatively for AMS. Striae are present in most parts of the cuticle of T, nativa. By contrast, the amorphous cuticle of T. pseudospiralis is not striated, it stains for carbohydrates and AMS. The cuticular surface of both Trichinella species is made up of a carbohydrate matrix visible only in cytochemical reactions. By contrast to the cuticle of T. pseudospiralis, the electron density of the cuticle of T. nativa is increased by transverse extensions of the outer, hypodermal membrane which contain numerous muscle tendons terminating in desmosomes. Muscle extensions together with lysosomes entering the hypodermis react for NE and cystine, but not for ACP. The number and configuration of hypodermal glands influences the ornamentation of the cuticle. A secretion released from these glands reacts for carbohydrates and proteins. Together with the carbohydrate coat known to be present on the surface of Trichinella species, they might bring about an immunity reaction in the host.

[Facts that explain the host specificity of helminths]. / Nekotorye fakty, ob''iasniaiushchie gostal'nuiu spetsifichnost' gel'mintov.

Extracts from larvae of Trichinella spiralis, mature Ascaridia galli and Ascaris suum and blood sera of their possible hosts (rats, pigs, chicks, guinea pigs, mice and men) were investigated by the method of electrophoresis in polyacrylamide gel. The protein component was found to be most identical in helminth and its obligate host. The host specificity is supposed to depend on the community of the protein content of the parasite and its host. The data on the difference in protein spectra are given and the peculiarities of the evolution of mono- and polyhost helminths are discussed.