A new procedure for marinating fresh anchovies and ensuring the rapid destruction of Anisakis larvae.

The consumption of marinated anchovies is the main route of transmission of anisakiasis in Spain. Because this country is one of the world's major tourist destinations, this traditional food also poses a potential health risk to millions of foreign visitors. Anisakis larvae are not destroyed by the traditional marinating procedure, and alternative methods, such as long-term storage in brine, freezing, or hydrostatic pressure treatment, all present major difficulties. In this study, we used high food-grade acetic acid concentrations (10, 20, 30, and 40% [vol/vol] in line with the quantum satis rule) to destroy these larvae rapidly, and we report data on the survival of Anisakis larvae exposed directly to different marinades and when the larvae are placed under the fish musculature. The percentage of salt and acetic acid in the fish tissue water phase was also determined. A marinating procedure is proposed that ensures the rapid death of Anisakis through the use of strong acetic acid concentrations. Posttreatment washes with water reduce these to levels acceptable to consumers. The sensory characteristics of the product were shown to be satisfactory. The actual selection of an acetic acid concentration for marinating depends on costs and the processing time available. The physiological stress of the larvae exposed to the different marinades was determined by measuring the levels of their stress proteins. The latter are good indicators of injury and might reflect the infectivity of larvae. In addition, we also used a rat model to determine the infectivity of larvae considered microscopically dead.

Oral inoculation with Gymnorhynchus gigas induces anti-parasite anapyhylactic antibody production in both mice and rats and adverse reactions in challenge mice.

This study was performed to mimic human consumption of fish flesh infected with larvae of the fish cestode Gymnorhynchis gigas and examine possible side effects thereof. Both a rat and a mouse G. gigas oral inoculation model were used. The rat model was evaluated according to propensity to induce stress responses in three tissues and anaphylactic antibody production. The mouse model measured anti-G. gigas IgG, M and A (H + L) levels in intestinal fluids, fecal suspensions and serum and specific serum IgE levels by enzyme-linked immunosorbent assay. Additionally, biological activity of anaphylactic antibodies in test mice and rats were evaluated utilizing challenge reinoculation(s) and intradermal skin testing, respectively. With the rat inoculation model, we noted both occurrence of a shock response, viz. increased expression of heat shock proteins in intestine and spleen, and of immediate-type skin reactions. No positive wheals were seen on skin sites treated with PBS or soluble Trichinella spiralis extract. With the mouse model, our results showed that all body fluids tested had significantly more anti-G. gigas IgG, M and A (H + L) than their counterparts from either PBS-treated or T. spiralis-infected controls. In addition, the mouse G. gigas model had significantly higher specific serum IgE. When challenged by oral route all test mice (n = 5) manifested immediate-type signs of distress. Repeated exposure to the "allergen", produced clinical signs appearing more rapidly and persisting longer. These findings suggest that feeding on fish infected with G. gigas plerocercoids triggers the production of anaphylactic-type antibodies in both rats and mice and, by implication, possibly also in humans.

Purification and preliminary characterization of a protease from the excretion-secretion products of Trichinella spiralis muscle-stage larvae.

A protease from excretion-secretion products of Trichinella spiralis muscle-stage larvae was purified by continuous elution electrophoresis. The state of purification was analyzed electrophoretically using one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme was shown to be a single polypeptide with an estimated molecular mass of 35 kDa and isoelectric point of 6.2. Following purification, the enzyme activity was measured by hydrolysis of gelatin, azocoll, azoalbumin, azocasein and collagen as substrates. Maximal azocollytic activity was at pH 5 and a temperature of 37 degrees C. Finally, the proteolytic activity was partially inhibited by N-alpha-p-tosyl-lysine chloromethyl ketone, chymostatin and E-64.

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).

Proteolytic enzymes from Trichinella spiralis larvae.

Trichinella spiralis larvae infect their hosts by the penetration of small intestine enterocytes. The exact mechanism of penetration is unknown, but the presence of proteolytic enzymes is suspected. In this study, whole worm extracts and excretory-secretory (ES) components were obtained and their proteolytic enzymes examined. Enzymes from worm extracts were capable of hydrolysing azocoll, a general protease substrate in a wide range of pH (2-8), with maximal activity at pH 5. Trichinella spiralis larval enzymes were sensitive to metalloprotease and serine protease inhibitors. Three proteases were identified in worm extracts at molecular weight (MW) 48, 54 and 62 kDa by incorporating a gelatine substrate into a standard or a modified sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) set-up, in which we used low SDS concentration in gel and electrophoresis buffer (0.01%). Intact larvae incubated in a medium containing azocoll showed azocollytic activity. Subsequent analysis of ES products by modified SDS-PAGE in gels containing gelatine demonstrated the presence of three protease of apparent MW 33, 62 and 230 kDa.

A study of proteases throughout the life cycle of Trichinella spiralis.

In the present report we study the proteolytic activity of the excretion-secretion and crude extracts of different stages of Trichinella spiralis (Owen, 1835) Railliet, 1895, (muscle-stage larvae, adult worms before and after mating, and newborn larvae) using natural substrates (structural and hematic mammalian proteins). The analysis of the results allow us to set up a certain stage-specificity, as well as an important relationship between the protease patterns throughout the parasite life cycle and how the parasite may overcome both mechanical and humoral barriers within the host. Muscle-stage larvae present a great activity against structural proteins (collagen), while newborn larvae and adult worms degrade principally hematic proteins (hemoglobin, fibrinogen and immunoglobulin G).

Presence of cholinesterases in Echinococcus granulosus protoscolices.

Cholinesterases were detected in protoscolices of Echinococcus granulosus spectrophotometrically and electrophoretically. To characterize these activities as acetylcholinesterases or pseudocholinesterases, BW284C51 and the organophosphate anthelmintic Neguvón were assayed as specific inhibitors of acetylcholinesterases, while Iso-OMPA was employed as specific inhibitor of pseudocholinesterases. We concluded that these cholinesterase (ChE) activities would be considered as possible targets in chemotherapy.

Detection of acetylcholinesterase activity and gamma-aminobutyric acid binding sites in Dicrocoelium dendriticum.

In the present study we report the presence of acetylcholinesterase activity and gamma-aminobutyric acid binding sites in crude extracts of Dicrocoelium dendriticum. This indirectly demonstrates the presence of acetylcholine and GABA. The presence of these neurotransmitters could indicate the existence of two systems implicated in the neurotransmission of the Digenea.

Comparison of cholinesterase activities in the excretion-secretion products of Trichinella pseudospiralis and Trichinella spiralis muscle larvae.

The presence of cholinesterases (ChE) is reported in T. pseudospiralis excretion-secretion products (ESP) by spectrophotometric method, using acetylthiocholine (ATCI) and butyrilthiocholine (BTCI) as substrates. By inhibition assays, we found that T. pseudospiralis release both acetyl- and butiryl-cholinesterases (AchE and BchE, respectively). The sedimentation coefficientes of these enzymes were determined by sucrose density gradient. We studied the in vivo ChE secretion by immunoblot assays using AchE from Electrophorus (electric eel) and sera from normal or infected mice with T. pseudospiralis or T. spiralis. The presence of anti-AchE antibodies was only demonstrated in the sera from T. pseudospiralis infected mice. Moreover the in vivo secretion was corroborated by the high difference determinate between the ChE activity of the immuno complexes from T. pseudospiralis infected sera and the immunocomplexes from T. spiralis infected sera as well as normal sera. Finally, we analyzed the effect of the organophosphate Neguvón (metrifonate) on the ChE activity from the T. pseudospiralis ESP. The drug inhibits in part this activity. Moreover Neguvón (metrifonate) showed a high activity against the T. pseudospiralis viability.

Freezing infective-stage larvae from Anisakis simplex and their produce at -20 degrees C for 24 h does not prevent the occurrence of autonomic imbalance in rat ileum.

UNLABELLED: In the present report, we analyze the efficacy of the sanitary regulations to kill the Anisakis simplex larvae (As L3) (heat or freeze) to avoid the gastrointestinal alterations that it provokes. We studied the effects on intestinal contractility (muscular tone, amplitude, and frequency of the twitches and cholinergic and alpha-adrenergic stimulus) of As L3, their crude extracts (CE) and excretory-secretory products (ESP), untreated or heated (60 degrees C for 15 min) or frozen (-20 degrees C for 24 h) using rat ileum and an isometric system. Carbachol and noradrenaline have been used as cholinergic and alpha-adrenergic stimulus, respectively. We determined that viable As L3, their untreated CE and ESP, as well as all their frozen counterparts, altered the intestinal contractile activity and its autonomic control. In contrast, heated As L3, CE, and ESP were incapable of provoking any change in rat ileum motility, suggesting an inhibitory effect by the heating procedure. IN CONCLUSION: 1) The gastrointestinal alterations provoked by As are not necessarily associated with a prior infection; and 2) the storage of the seafood at -20 degrees C during 24 h does not prevent the intestinal autonomic imbalance provoked by As, whereas, the prior heating at 60 degrees C for 15 min may completely prevent such process.

A 24-kDa collagenase from Gymnorhynchus gigas elicits rat ileum hyperreactivity and is a target of humoral responses in mice previously given a single oral dose of parasite extract.

Fish declared fit to be eaten may contain plerocercoids (larvae) of the fish cestode Gymnorhynchus gigas. We showed previously that crude G. gigas larval extract given in a once-only oral dose to either mice or rats induces parasite-specific immediate-type responses and that this extract evokes increased contractile activity in normal rat ileums. We show here that a 24-kDa collagenase (24-kCol), purified from the crude extract is (1) a target of both local (intestinal) and systemic IgE responses in mice sensitized by oral G. gigas and (2) elicits considerable changes in rat ileum contractility. Exposure of rat ileum segments once to 7 microg 24-kCol significantly increased tone and amplitude, but not frequency, of contractions compared with control recordings. In all, these studies have indicated 24-kCol, an abundantly produced protein of G. gigas larvae, to be a participant in potentially serious/adverse intestinal responses in both mice and rats. Such responses are very likely to occur in "sensitized" humans also.

Antibody response to a protease secreted by Trichinella spiralis muscle larvae.

In the present study we analyzed the humoral response of Trichinella spiralis-infected mice to a 35-kDa protease (purified from the excretory-secretory products of T. spiralis muscle larvae) by a Western-blot procedure and an enzyme-linked immunosorbent assay (ELISA) technique using a panel of postinfection mouse anti-Trichinella sera. The results demonstrated that this response was time-dependent and that infected mice could be distinguished from controls. In addition, inhibition assays demonstrated that these antisera were capable of abolishing the proteinase activity of the 35-kDa protease in vitro. The occurrence of proteases seems to be a very common feature in parasite crude extracts and excretory-secretory products (McKerrow 1989). It is also known that these enzymes are implicated in important host-parasite interactions, and for this reason, recent reports have proposed the use of parasite proteases both as alternative targets for an induced immune response and as a rich source of antigenic material for diagnostic testing (Hotez et al. 1985; Yamasaki et al. 1989; Song et al. 1990; Frank and Grieve 1991; Britton et al. 1992; Song and Chappell 1993). We have recently purified a protease (mol. wt., 35 kDa) from the excretory-secretory (ES) products of Trichinella spiralis (GM-1 strain) muscle larvae and established some of the biochemical properties of this protease (Armas-Serra et al. 1994).(ABSTRACT TRUNCATED AT 250 WORDS)

Proteolytic activity of the Gymnorhynchus gigas plerocercoid: purification and properties of a collagenase from the crude extract.

The present report demonstrates that the Gymnorhynchus gigas plerocercoid possesses various types of endo- and exoproteases with activity against general (azocoll, azocasein, and azoalbumin) and specific substrates (elastin, keratin, collagen, hemoglobin, fibrinogen, plasma, and immunoglobulin G). The activity against collagen is principally due to a 24-kDa collagenase with an isoelectric point of 7.5 and without isoforms or sugar residues. Moreover, its high degree of proteolytic activity against collagen under conditions similars to those encountered by the parasite in its hosts (pH and temperature) and its similarity to metallo- and cysteine proteases (the principal protease types implicated in degradation of tissues) suggests the importance of this molecule as a lytic enzyme principally implicated in penetration processes across the teleost muscle or/and into the gastrointestinal system of elasmobranch fishes as well as in molting processes.

Purification and partial characterization of proteinase activity in eggs of Dicrocoelium dendriticum.

A preliminary purification has been carried out by continuous elution electrophoresis of a 49.5 kDa protease of crude extracts from Dicrocoelium dendriticum eggs. The enzyme showed a high capacity to degrade the collagen derivative azocoll at acidic pH. Although it is necessary to carry out further experiments to confirm any physiological role, this protease could be implicated in penetration mechanisms.

Altered autonomic control in rat intestine due to both infection with Anisakis simplex and incubation with the parasite's crude extract.

Anisakis simplex IgE may bring on allergic responses such as angioedema, vomiting, and urticaria from eating seafood, but it is not the only etiology. Induced cholinergic hyperreactivity or adrenergic blockade in the target tissue can cause these diseases nonimmunologically also. Here we studied the effects on normal intestinal motility of brief A. simplex infections and in vitro exposures to the parasite's extract (CE). Each approach was evaluated according to its ability to induce cholinergic hyperreactivity or adrenergic blockade in rat duodenum (RD), jejunum (RJ), and ileum (RI) in vitro. Additionally, bolus propulsion in RD, RJ, and RI was evaluated with time in vivo utilizing animals infected 4 h previously with A. simplex larvae (L3) vs sham animals. Tissues, after inoculation of 1, 5, 10, and 20 L3, exhibited time- and dose-dependent motility changes after carbachol (Ch) and noradrenaline (NA), justifying our using herein rats from the fourth hour of infection with 20 L3. We observed a persistent, yet differential effect of the infection on RD, RJ, and RI responses to Ch or NA. It caused cholinergic (muscarinic) hyperreactivity in RD only, and adrenergic blockade in all other parts, and consequently increased the transit index in RD, not in RJ or RI. In contrast, exposing RD, RJ, and RI to CE persistently increased both parameters, amplitude of twitches and muscular tone, in all, albeit that, here also, responses to Ch and NA were CE dose dependent. Interestingly, sensitivity to CE was in the order RI > RJ > RD, the reverse situation of that observed during active infection. Thus, previously viable A. simplex L3, after digestion, can exert bystander disturbance in autonomic control in the whole intestine. Our findings demonstrate that A. simplex L3, alive or dead, can induce cholinergic hyperactivity and adrenergic blockade in the whole small intestine and, as a consequence, gastrointestinal symptoms. Significantly, they may do so long before parasite-specific IgE is detectably induced or despite the occurrence of such IgE.

Localization of a 24-kDa collagenase in the Gymnorhynchus gigas plerocercoid.

A 24-kDa collagenase was localized in the Gymnorhynchus gigas plerocercoid immunohistochemically by peroxidase complex staining using polyclonal antibodies from NMRI mouse sera immunized with purified enzyme. Immunoreactivity was determined at different parts of the body (scolex, vesicle and caudal region) and mainly localized in microtriches and parenchymal tissues of the scolex and vesicle. These results, along with the absence of the enzyme in the plerocercoid excretion-secretion products, suggest that the 24-kDa collagenase is produced by parenchymal cells in the anterior region and transported to the outer regions of the worm It is possible that the enzyme plays an important role in degrading parasite tissues during the moulting process.

Biochemical effects of luxabendazole on Trichinella spiralis.

Biochemical changes produced by luxabendazole in muscle-stage Trichinella spiralis larvae consisted of a decrease in free glucose and glycogen levels (46.71% and 35.66%, respectively) after in vivo treatment, slight in vitro inhibition of fumarate reductase activity (24.15%) and, finally, inhibition of [3H]-colchicine-tubulin binding, which was found to be of a competitive nature, with an inhibition constant (Ki) of 0.9 x 10(-7) M. In a parallel study, luxabendazole did not appear to be inhibitory to [3H]-colchicine binding to pig-brain tubulin.