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.

Relationship between heat shock protein levels and infectivity in Trichinella spiralis larvae exposed to different stressors.

The aim of the present study was to investigate the relationship between infectivity and the levels of two major heat shock proteins (Hsp70 and Hsp60) in Trichinella spiralis larvae. Parasites were exposed to either sublethal thermal stress (43 and 45 degrees C) or to warm or cold temperature oxidative stress. The stressed larvae were then inoculated into female CD1 mice to determine their infectivity. Hsps were detected and quantified by Western blotting using monoclonal antibodies. Infectivity was expressed as larvae per gram of muscle. Warm temperature oxidative stress (20 mM H2O2 at 37 degrees C) caused a significant increase in Hsp levels and total loss of infectivity. Cold oxidative stress (20 mM H2O2 at 4 degrees C) caused no alterations in either Hsp levels or infectivity. However, high oxidative stress and cold (200 mM H2O2 at 4 degrees C) caused a slight increase in Hsp60 levels and a drastic reduction in infectivity. Exposure of the larvae to 43 or 45 degrees C did not significantly alter Hsp levels or infectivity. These results show that (i) cold reduces the deleterious effects of oxidative stress; (ii) heat induces neither increased Hsp60/Hsp70 levels nor reduces infectivity; (iii) increased Hsp levels induced by oxidative stress may cause lower infectivity.

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.

Heat shock protein synthesis over time in infective Trichinella spiralis larvae raised in suboptimal culture conditions.

Changes in the viability, infectivity and heat shock protein (Hsp) levels are reported in Trichinella spiralis first stage larvae (L1) stored in 199 medium for up to seven days at 37 degrees C. These conditions induce stress that the larvae, eventually, cannot overcome. After three days of storage, the infectivity and viability were unchanged, although higher Hsp70 levels were observed. After this time, larvae gradually lost viability and infectivity, coinciding with a decrease in Hsp70 and Hsp90 and an increase in actin (a housekeeping protein). In addition, a possibly inducible heat shock protein, Hsp90i, appeared as constitutive Hsp90 disappeared. No significant changes in Hsp60 levels were detected at any time. These results suggest that heat shock proteins initially try to maintain homeostasis, but on failing, may be involved in cell death.

Cholinesterase and phosphatase activities in adults and infective-stage larvae of levamisole-resistant and levamisole-susceptible isolates of Haemonchus contortus.

Cholinesterase (ChE) and acid phosphatase (AP) activities, but not alkaline phosphatase activities, were detected in cytosolic and membrane-bound fractions of adult and infective-stage larvae of levamisole-resistant and levamisole-susceptible Haemonchus contortus. In contrast to other gastrointestinal nematodes, the ChE activity was higher in L3 than in adults and, in both cases, was mainly associated with membranes. ChE activity was inhibited by Triton X-100 and was only detected in membrane-bound fractions when the detergent was removed. Differences between resistant and susceptible L3 were observed in the response to inhibitors (cytosolic fraction) and in the enzymatic content (membrane-bound fraction). Phosphatase activity was detected at acidic pH in all fractions, being higher in the adult than in the L3 stage. In the former, most of the enzyme was localized in the membrane-bound fractions, whereas in the latter it was mainly in cytosolic fractions. This difference could be correlated with the activity in the gut. In inhibition assays, a difference between cytosolic fractions from resistant and susceptible adults was observed in their response to 1 mmol/L tartaric acid.

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.

Expression of Hsp90, Hsp70 and Hsp60 in Trichinella species exposed to oxidative shock.

Stress response and phosphorylation of heat shock proteins (HSPs) 60, 70 and 90 were studied in Trichinella nativa, T. nelsoni, T. pseudospiralis and T. spiralis larvae at 30-min intervals following exposure to 20, 100 and 200 mM H2O2. There was a time- and dose-dependent differential survival for the infective stage larvae (L1) of these four Trichinella species. Immunoblotting analysis revealed that constitutive Hsp60 and Hsp70, but not Hsp90, from test Trichinella species are constitutively phosphorylated on serine/threonine residues as they converted to forms with increased sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) mobility by treatment with alkaline phosphatase. After exposure to H2O2, while there was a time-related occurrence of the three HSPs with decreased SDS-PAGE mobility, these HSPs were insensitive to alkaline phosphatase except in the case of exposure to 20 mM H2O2 for Hsp60 from all Trichinella species and Hsp70 from T. spiralis and T. nelsoni. The synthesis of HSPs forms with decreased SDS-PAGE mobility is a susceptibility signal because the lower concentration of peroxide (20 mM) did not cause a decrease on HSPs SDS-PAGE mobility in T. spiralis and T. nelsoni, the two more resistant selected Trichinella species.

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.

Could ivermectin have a synergic effect with albendazole in hydatidosis therapy?

The efficacy of a combination of ivermectin (IVM) and albendazole (ABZ) used as a chemoprophylactic treatment and as treatment of secondary hydatidosis is described for the first time. IVM treatment alone was not effective against Echinococcus granulosus, either when the protoscolices were recently inoculated or when they had developed to the metacestode stage. However, the efficacy of IVM and ABZ when used in combination as a prophylactic treatment was 95.72% and 87% with respect to the number and the wet weight of cysts, respectively. These results were higher than in the treatment of secondary hydatidosis, which were 44.8% and 45.26%, respectively. The ultrastructural changes in the germinal layer of the cysts after the treatments are also described.

Oxidative and cold shock cause enhanced induction of a 50 kDa stress protein in Trichinella spiralis.

The anti-heat shock protein (Hsp)-90 monoclonal antibody AC-16 reacts on blots with Hsp90 and a 50 kDa protein (prot-50) from infective-stage (L1) larvae of the nematode Trichinella spiralis. We examined Hsp90 and prot-50 levels by densitometric analysis of immunoblots of T. spiralis larval extracts prepared before (time 0, 37 degrees C) and after oxidative [hydrogen peroxide (H2O2)] stress, or cold shock at 4 degrees C. Extracts from H2O2-exposed L1 were obtained after 2 h; the others at 2, 4, and 8 h after the temperature shift. After H2O2 shock, the constitutive Hsp90 and prot-50 were both significantly induced and appeared as slower migrating inducible isoforms. However, whereas Hsp90 levels decreased after cold shock, prot-50 levels immediately and persistently increased after shock at 4 degrees C. These data present compelling evidence that the prot-50 described here functions as a Hsp and a cold shock protein.

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.

Could ivermectin have a synergic effect with albendazole in hydatidosis therapy?

The efficacy of ivermectin (IVM) alone, albendazole (ABZ) alone and a combination of both (IVM + ABZ) against Echinococcus granulosus protoscolices was studied by means of in vitro incubation. The maximum protoscolicidal effect was detected when we used IVM+ABZ in combination. In this case, protoscolex viability dropped to 35% in comparison with 50% for IVM alone and 82.5% with ABZ alone 18 days post-incubation. Only the protoscolices incubated with IVM + ABZ did not develop into cysts following their inoculation into mice. The changes in ultrastructure induced in the protoscolices after the different drug incubations are also described. The incubation of cysts with IVM and IVM + ABZ shows that IVM + ABZ in combination is more effective than IVM alone as only when we incubated the cysts with IVM + ABZ did they completely lose their infectivity to mice. This occurred after only 10 days post-incubation. Cellular alterations were also more marked with IVM+ABZ incubations with: (1) the presence of residual bodies, (2) numerous lipids droplets and (3) vacuoles in the cytoplasm of cytons. However, the truncated microtriches and the nuclei remained unaltered after 10 days.

Ultrastructural alterations induced by nifurtimox and another nitro derivative on epimastigotes of Trypanosoma cruzi.

We report the ultrastructural alterations induced on epimastigotes by nifurtimox and 5-nitro-2-thienyl-malononitrile (5NO2TM), a novel compound with anti-Trypanosoma cruzi activity. Parasites treated with concentrations of nifurtimox lower than usually employed for this kind of study showed vacuolisation, alterations of the mitochondria, the nucleus and the ribosomes. 5NO2TM caused the same kind of damage, but to a greater degree. This result correlates with the fact that cultures treated with this compound experienced a greater loss of viability.

Relationship between the efficacy of praziquantel treatment and the cystic differentiation in vivo of Echinococcus granulosus metacestode.

The cyst differentiation of protoscolices inoculated in NMRI mice was studied in detail. Although the laminated layer began its formation at 21 days post-infection (p.i.), we could not identify this structure in all parasites until 34 days p.i.. At 34 days p.i., all protoscolices were transformed into the metacestode stage. Subsequently, the efficacy of praziquantel (PZ) was studied in different stages of development of the parasite. We have found a different PZ susceptibility in both earlier stages and well-developed cyst. PZ treatment exhibited a high efficacy at the beginning of cyst differentiation and a zero efficacy in the mature metacestode when the cystic layers were totally developed. The relationship between the tegumental ultrastructural changes occurring during the vesicular evolution of protoscolices in NMRI mice and the efficacy of PZ treatment is discussed in the present study.

Reacciones cruzadas entre Anizakis simplex y Gymnorhynchus gigas: estudio experimental en ratones NMRI / Cross reactions between Anisakis simplex and Gymnorhynchus gigas: experimental study in NMRI mice

En el presente trabajo se ha realizado el seguimiento de la respuesta inmunitaria humoral que provoca la ingesta de diferentes fracciones y dosis de la larva plerocercoide de Gymnorhynchus gigas, utilizando ratones NMRI como modelo de laboratorio. Para ello y mediante la técnica de ELISA se han determinado los niveles de IgG, M, A y E en suero y mucosa intestinal. Asimismo, se han analizado las posibles reacciones cruzadas que se puedan producir con la L3 de Anisakis simplex, ensayando las reacciones de cada suero y cada mucosa tanto frente a antígenos somáticos, como frente a antígenos de excreción-secreción de la L3 de A: simplex y de la larva plerocercoide de G. gigas, según procedía. Los resultados obtenidos indican que I) G. gigas provoca una respuesta inmunitaria humoral tanto a nivel general como entérico, al producirse un aumento de las Ig en ambos niveles; II) los dos parásitos comparten antígenos, ya que existen reacciones cruzadas entre ambos, y III) esos antígenos actúan como alergenos, al ser capaces de producir un aumento de las IgE. (AU)

Depolarization of the tegument precedes morphological alterations in Echinococcus granulosus protoscoleces incubated with ivermectin.

The nematocidal activity of ivermectin (IVM) largely arises from its activity as a potent agonist of muscular and neuronal glutamate-gated chloride channels. A cestocidal effect has also been suggested following in vitro treatments, but the molecular basis of this activity is not clear. We studied the effect of IVM on the metacestode stage of the tapeworm Echinococcus granulosus by assessing the viability, ultrastructure, and tegumental membrane potential as a function of drug concentration and incubation time. Concentrations of 0.1 and 1.0 microg/ml of IVM had no effect on any of these three parameters for up to 6 days of treatment. A concentration of 10 microg/ml, however, elicited a sequence of alterations that started with a approximately 20-mV depolarization of the tegumental membrane, and was followed by rostellar disorganization, rigid paralysis and, eventually, loss of viability. It is likely that the IVM-induced depolarization of the tegument acts as the signal that initiates the cascade of degenerative processes that leads to the parasite's death. This would place the tegument as the primary target of action of IVM on cestodes. As an appropriate chemotherapy for the hydatid disease is still lacking, the cestocidal effect of IVM reported here is worth considering.

Combined praziquantel and albendazole chemoprophylaxis in experimental hydatidosis.

The results of the chemoprophylaxis of experimental hydatidosis with a combination of praziquantel and albendazole are presented. In vivo studies were performed on NMRI mice injected with protoscolices of Echinococcus granulosus. The treatment was the same as that previously used by us, but reducing the treatment period to only 1 month. Four months after injection , the mice were killed and autopsied. Developed hydatid cysts were counted, weighed and processed for observation by transmission electron microscopy. The efficacy of the present treatment was 97.7% and 97.15%, in number and weight of the cysts respectively, only slightly lower than that obtained by us after 4 months of treatment. Moreover, the few cysts that had developed in the treated mice showed severe damage to their ultrastructure.

In vitro effects of anti-Trypanosoma cruzi compounds on HSP60 levels.

The effect of nifurtimox and 5-nitro-2-thienylmalononitrile (5NO2TM), a novel compound with anti-Trypanosoma cruzi activity, upon vitality and HSP60 immunoreactivity of epimastigotes, has been determined. Both products showed no activity against epimastigotes at 0.1 microg/ml, while at 0.5 and 1 microg/ml, after 24 h of incubation, densities of these groups were significantly reduced, when compared to controls. An enhancement of HSP60 immunoreactivity was observed after 24 h in groups treated with 0.5 and 1 microg/ml nifurtimox. On the other hand, 5NO2TM had no effect.

Physiological and haematological consequences of a novel parasite on the red-rumped swallow Hirundo daurica.

Parasite virulence has been hypothesised to increase with the degree of host sociality because highly social hosts have a greater probability of encountering horizontal transmission of parasites and experiencing infections with multiple strains of the same parasites than do solitary hosts. As compared with the defences of closely related social host species, we predicted that solitary hosts should have relatively weak defences against parasites, thus being relatively more affected when parasitised by a novel parasite. We tested this prediction by either experimentally infesting 12 nests of the solitarily nesting red-rumped swallow Hirundo daurica with 50 individuals of the generalist martin bug Oeciacus hirundinis or by fumigation of nine nests. Nestlings 13 days old from the parasite addition group experienced increased mortality, attained lower body mass and tended to have shorter tarsi compared to nestlings from fumigated nests. Surprisingly, nestlings from the parasite addition group had higher packed cell volume (cellular fraction of blood) and lower levels of heat shock proteins (HSP60) than nestlings from the fumigation group. A measure of immunocompetence was not significantly affected by treatment, but its magnitude was positively related to packed cell volume and negatively related to level of HSP60. Solitary hosts like the red-rumped swallow have weak immune responses and low levels of heat shock proteins when infested with ectoparasites while highly social hosts have strong immune responses and high levels of heat shock proteins when infested. These findings partially support the hypothesis that potential host species with weak defences are more susceptible to infection and the deleterious effects of evolving parasites than potential hosts with strong defences.