Francisco Javier Adroher

The fishing area as a possible indicator of the infection by anisakids in anchovies (Engraulis encrasicolus) from southwestern Europe.

A study was conducted on the parasitization by anisakids of European anchovies (Engraulis encrasicolus) from the Eastern Atlantic (Gulf of Cádiz and Strait of Gibraltar) and Western Mediterranean (Ligurian Sea, Gulf of Lion, Catalonia coast and NW Alborán Sea) throughout 1998 and 1999. The anisakids detected were identified as third larval stages of Anisakis larva type I and Hysterothylacium aduncum. Global prevalence was 9.4% for Anisakis and 24.5% for H. aduncum. Analysis of the origin of the anchovies revealed a higher prevalence of Anisakis than H. aduncum in fish from the Atlantic and vice-versa in fish from the Mediterranean. Analysis of various fishing areas in the Western Mediterranean revealed a prevalence of Anisakis in fish from the Ligurian Sea that was 5-fold or more than in the other three areas, with a significantly greater prevalence of H. aduncum in fish from the NW Mediterranean than from the Spanish Mediterranean coast. The prevalence of infection was found to be significantly related to anchovy length for both Anisakis and H. aduncum. More than 55% of Anisakis larvae were found in the muscle. According to these data, the risk of acquiring anisakiasis/anisakidosis from the consumption of raw or under-cooked anchovies may depend upon the area in which they were caught.

Larval anisakids (Nematoda: Ascaridoidea) in horse mackerel (Trachurus trachurus) from the fish market in Granada (Spain)

Abstract Horse mackerel (Trachurus trachurus) from the fish market in Granada, southern Spain, were surveyed for anisakid nematodes. The fish came from fishing ports all over the country. Larval anisakids were found in 39.4% of the fish examined; 26.1% were infected with third-stage larvae (L3s) of Anisakis simplex; 0.3%, with A. physeteris L3s; 31.1%, with Hysterothylacium aduncum L3s; and 1.7%, with fourth-stage larvae (L4s) of Hysterothylacium sp. Horse mackerel from Mediterranean Sea coastal ports (South and Southeast Spain) had the lowest levels of infection, and those from Cantabrian Sea coastal ports (North Spain) had the highest. The variation in infection levels with host size (age) and season of capture are surveyed.Abstract Horse mackerel (Trachurus trachurus) from the fish market in Granada, South Spain, were surveyed for anisakid nematodes. The fish came from fishing ports all over the country. Larval anisakids were found in 39.4% of the fish examined. In all, 26.1% were infected with third-stage larvae (L3s) of Anisakis simplex; 0.3%, with A. physeteris L3s; 31.1%, with Hysterothylacium aduncum L3s; and 1.7%, with fourth-stage larvae (L4s) of Hysterothylacium sp. Horse mackerel from Mediterranean Sea coast ports (South and Southeast Spain) had the lowest levels of infection and those from Cantabrian Sea coast ports (North Spain) had the highest levels. Variations in infection levels with host size (age) and season of capture were surveyed.

Larval anisakids parasitizing the blue whiting, Micromesistius poutassou, from Motril Bay in the Mediterranean region of southern Spain

A total of 301 blue whiting, Micromesistius poutassou Risso, 1826, ranging in length from 17 to 28 cm, from Motril Bay (Mediterranean coast, south Spain) were examined for anisakid nematodes, as these fish are common items in the Spanish Mediterranean diet. Three anisakid species were morphologically identified with a total prevalence of 10.63%. Anisakis simplex s.l. Rudolphi, 1809 had a prevalence value of 6.65%, compared with 2.66% for A. physeteris Baylis, 1923 and 2.33% for Hysterothylacium aduncum Rudolphi, 1802. Variations in prevalence values with season and host size are discussed. Allozyme markers (leucine aminopeptidase-1) were used to identify anisakid nematodes assigned to the A. simplex complex and all examined larvae were found to correspond genetically to A. pegreffii Nascetti et al., 1986.

Nested polymerase chain reaction for detection of Theileria annulata and comparison with conventional diagnostic techniques: its use in epidemiology studies.

Abstract In this work we studied the ability of a nested polymerase chain reaction (PCR) to detect Theileria annulata, the causative agent of Mediterranean theileriosis, in blood samples obtained from cattle on farms in different Spanish regions and its possible use in epidemiology studies. Of the 214 samples analyzed, 78.04%, 69.86%, and 62.26% were found to be positive by nested PCR, indirect immunofluorescent antibody test, and optical microscopy of Giemsa-stained smears, respectively. The three techniques were in agreement in 68.6% of the results. The observation that the prevalence of Mediterranean theileriosis estimated using nested PCR alone (70.3%) and that obtained using all three diagnostic techniques together (80.4%) did not significantly differ verifies the utility of this technique in epidemiology studies.

Influence of electrolytes and non-electrolytes on growth and differentiation of Trypanosoma cruzi

The influence of electrolytes and non-electrolytes, especially NaCl and sorbitol, on the metacyclogenesis and growth of Trypanosoma cruzi has been studied. The addition of 50 or 100 mEq/l NaCl to the culture media significantly increased the development of metacyclic forms. Other electrolytes and non-electrolytes had no effect on epimastigote-metacyclic differentiation. The growth rate was never modified to any extent. The influence of sodium concentration, osmotic pressure, among other factors, are discussed. Electrophoresis showed proteins bands which could be related either to the adaptation of T. cruzi to the new culture media or to the initiation of differentiation processes.

Differential energetic metabolism during Trypanosoma cruzi differentiation: I. Citrate synthase, NADP-isocitrate dehydrogenase, and succinate dehydrogenase

The activities of the mitochondrial enzymes citrate synthase (citrate oxaloacetatelyase, EC 4.1.3.7), NADP-linked isocitrate dehydrogenase (threo-Ds-isocitrate:NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42), and succinate dehydrogenase (succinate: FAD oxidoreductase, EC 1.3.99.1) as well as their kinetic behavior in the two developmental forms of Trypanosoma cruzi at insect vector stage, epimastigotes and infective metacyclic trypomastigotes, were studied. The results presented in this work clearly demonstrate a higher mitochondrial metabolism in the metacyclic forms as is shown by the extraordinary enhanced activities of metacyclic citrate synthase, isocitrate dehydrogenase, and succinate dehydrogenase. In epimastigotes, the specific activities of citrate synthase at variable concentrations of oxalacetate and acetyl-CoA were 24.6 and 26.6 mU/mg of protein, respectively, and the Michaelis constants were 7.88 and 6.84 microM for both substrates. The metacyclic enzyme exhibited the following kinetic parameters: a specific activity of 228.4 mU/mg and Km of 3.18 microM for oxalacetate and 248.5 mU/mg and 2.75 microM, respectively, for acetyl-CoA. NADP-linked isocitrate dehydrogenase specific activities for epimastigotes and metacyclics were 110.2 and 210.3 mU/mg, whereas the apparent Kms were 47.9 and 12.5 microM, respectively. No activity for the NAD-dependent isozyme was found in any form of T. cruzi differentiation. The particulated succinate dehydrogenase showed specific activities of 8.2 and 39.1 mU/mg for epimastigotes and metacyclic trypomastigotes, respectively, although no significant changes in the Km (0.46 and 0.48 mM) were found. The cellular role and the molecular mechanism that probably take place during this significant shift in the mitochondrial metabolism during the T. cruzi differentiation have been discussed.

In vitro cultivation of Anisakis simplex: pepsin increases survival and moulting from fourth larval to adult stage

This paper describes the in vitro cultivation of the 3rd-larval stage (L3) of Anisakis simplex to adulthood in a much simpler and easier to prepare medium than those described to date. The adult males obtained are between 3.8 and 6.5 cm long and the females between 4.5 and 8.0 cm. Some individually cultivated females laid eggs which had an average size of 44.4 x 50.5 microm. The culture conditions were as follows: medium RPMI-1640 supplemented with 20% heat-inactivated fetal bovine serum and 1% commercial pepsin, at pH 4.0 and a temperature of 37 degrees C, and in air atmosphere with 5% CO2. The pepsin was found to be the key to the success of the culture. The average survival of the worms in the culture increased from 50 to 88 days, due to the fact that the survival of the adults practically doubled (increasing by 1.9 times). Furthermore, the number of worms that completed the 4th moulting (M4) increased by 4.2 times, from 22.9 to 95.6%. This culture medium may facilitate, due to its simplicity, the study of anisakids, or at least of A. simplex, constituting another step towards achieving a complete in vitro life-cycle for these parasites.

DIFFERENTIAL BEHAVIOUR OF GLUCOSE 6-PHOSPHATE DEHYDROGENASE IN TWO MORPHOLOGICAL FORMS OF TRYPANOSOMA CRUZI

1. Glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49) of two morphological forms of Trypanosoma cruzi, epimastigotes and metacyclics, are reported. 2. The kinetic behaviour and some of the kinetic parameters of the enzyme in both forms were studied. The enzymes showed a simple Michaelis-Menten kinetic. 3. The activity in epimastigote forms was alway higher than the metacyclic ones. At subsaturating concentrations of substrate was almost 10-fold higher, whereas at saturating concentrations was about 2-fold higher. 4. In epimastigote forms the specific activity and Km values, at pH 7.5 and 37 degrees C, was found to be 142 mUnits x mg-1 of protein and 0.23 mM, respectively. 5. In the same conditions, the specific activity and Km values in metacyclic forms was 75 mUnits x mg-1 of protein and 1.06 mM, respectively. 6. A possible role in the carbohydrate metabolism of glucose 6-phosphate dehydrogenase in both forms of Trypanosoma cruzi is discussed.

Differential energetic metabolism during Trypanosoma cruzi differentiation. II. Hexokinase, phosphofructokinase and pyruvate kinase

SummaryThe activities of hexokinase (ATP:hexose-6-phosphate transferase, E.C. 2.7.1.1), phosphofructokinase (ATP: fructose-6-phosphate 1-phosphotransferase, E. C. 2.7.1.11) and pyruvate kinase (ATP: pyruvate transferase, E.C. 2.7.1.40), and their kinetic behaviour in two morphological forms of Trypanosoma cruzi (epimastigotes and metacyclic trypomastigotes) have been studied. The kinetic responses of the three enzymes to their respective substrates were normalized to hyperbolic forms on a velocity versus substrate concentration plots. Hexokinase and phosphofructokinase showed a higher activity in epimastigotes than in metacyclics, whereas pyruvate kinase had similar activity in both forms of the parasite. The specific activity of hexokinase from epimastigotes was 102.00 mUnits/mg of protein and the apparent Km value for glucose was 35.4 μM. Metacyclic forms showed a specific activity of 55.25 mUnits/mg and a Km value of 46.3 μM. The kinetic parameters (specific activity and Km for fructose 6-phosphate) of phosphofructokinase for epimastigotes were 42.60 mUnits/mg and 0.31 mM and for metacyclics 13.97 mUnits/mg and 0.16 mM, respectively. On the contrary, pyruvate kinase in both forms of T. cruzi did not show significant differences in its kinetic parameters. The specific activity in epimastigotes was 37.00 mUnits/mg and the Km for phosphoenolpyruvate was 0.47 mM, whereas in metacyclics these values were 42.94 mUnits/mg and 0.46 mM, respectively. The results presented in this work, clearly demonstrate a quantitative change in the glycolytic pathway of both culture forms of T. cruzi.

Cathepsin B- and L-like cysteine protease activities during the in vitro development of Hysterothylacium aduncum (Nematoda: Anisakidae), a worldwide fish parasite.

Proteinases play an important role as virulence factors both in the life-cycle of parasites and in the pathogen-host relationship. Hysterothylacium aduncum is a worldwide fish parasite nematode which has been associated with non-invasive anisakidosis and allergic responses to fish consumption in humans. Cysteine proteinases have been associated with allergy to plant pollens, detergents and dust mites. In this study the presence of two types of cysteine proteinases (cathepsin B and cathepsin L) during in vitro development of H. aduncum is investigated. Specific fluorescent substrates were used to determine cathepsin activities. The activity detected with substrate Z-FR-AMC was identified as cathepsin L (optimum pH=5.5; range 3.5-6.5). Cathepsin B activity was only identified with Z-RR-AMC (optimum pH=7.0-7.5; range 5.0-8.0). The start of cultivation led to increased activity of both cathepsins (1.8-fold for cathepsin B and 6.3-fold for cathepsin L). These activities varied according to the developmental stage. Cathepsin B activity decreased after M4, returning to its initial level. Cathepsin L activity also decreased after M4, but still maintained a high level (4-6 times the initial level) in adult stages. Having considered these activity variations and the optimum pH values, we suggest that cathepsin L has a role in digestive processes while cathepsin B could be involved in cuticle renewal, among other possible functions.