Rocío Benítez

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.

Anisakis spp. in European hake, Merluccius merluccius (L.) from the Atlantic off north-west Africa and the Mediterranean off southern Spain

We studied the parasitization by Anisakis spp. in European hake (Merluccius merluccius) of 40–52 cm from the Atlantic off north-west Africa and the Mediterranean off southern Spain. Infection parameters differed: The fish from the Atlantic showing a prevalence of 87.97% and a mean intensity of 4.69, while, in those from the Mediterranean, these were 41.27% and 1.73, respectively. In both samples the two-third larval stage types were isolated: Anisakis simplex sensu lato and Anisakis larvae type II, with prevalence of 85.71% and 30.83% in fish from the Atlantic and 41.27% and 1.59% for those from the Mediterranean, respectively. In both samples, the prevalence of larvae in viscera was clearly higher than in the muscle tissue. We also observed an increase in parasitization with increasing host length, those ≥ 46 cm having the highest prevalences (94.87% for those from the Atlantic and 58.33% for those from the Mediterranean; p<0.03).

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.

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.

In vitro cultivation of Hysterothylacium aduncum (Nematoda: Anisakidae) from 3rd-stage larvae to egg-laying adults

This is the first demonstration of the in vitro development of the 3rd-stage larvae (L3) of Hysterothylacium aduncum to the adult. This was achieved in a semi-defined medium that is easy to prepare and to reproduce. The L3, collected from the peritoneal cavity of horse mackerel (Trachurus trachurus), were individually inoculated into RPMI-1640 medium +20% heat-inactivated fetal bovine serum (IFBS). It has been demonstrated that the optimum temperature for development is around 13 degrees C and is stimulated by the presence of 5% CO2 in the growth atmosphere, increasing the percentage moulting to the 4th larval stage (L4) by 1.9-fold (from 44 to 82%) and the average survival of the nematodes by 1.6 times (from 60 to 96 days). When the larvae were grown at different pHs, optimum development occurred at pH 4.0. Under these conditions, all the larvae moulted to the L4 and more than two-thirds transformed to the adult stage--in which 25-30% of the females laid eggs--and reached an average survival of over 4 months. When this medium was supplemented with 1% (w/v) of commercial pepsin, all the larvae reached the adult stage, at least 45% of the females oviposited, laying around 12-fold more eggs per female than in the medium without pepsin. The mean size of the eggs (non-fertilized) obtained was 56.8 x 47.6 microm. The mean length of the adult males obtained was between 3.2 and 5.2 cm and the females were between 3.0 and 6.5 cm. The adult specimens were morphologically identified as Hysterothylacium aducum aduncum. This culture medium (RPMI-1640+20% (v/v) IFBS+1 commercial pepsin, at pH 4.0, 13 degrees C and 5% CO2 in air) could facilitate the identification of at least some of the larvae of the genus Hysterothylacium--and perhaps other anisakids--for which the specific identification and the biological study of these parasites is often difficult.

Fishing area and fish size as risk factors of Anisakis infection in sardines (Sardina pilchardus) from Iberian waters, southwestern Europe

The sardine (Sardina pilchardus) is a fish commonly consumed and appreciated in many countries, although they are more likely to be eaten fresh in western Mediterranean countries such as Spain, Portugal, France or Italy. A molecular epidemiological survey of sardines from 5 fishing areas of the Spanish Mediterranean (Málaga, southern Spain) and Atlantic coasts (southern: Cádiz and Isla Cristina; northern: A Coruña and Ondarroa) was carried out to determine the presence of Anisakis spp. larvae. The highest prevalence of these larvae was observed in fish from A Coruña (28.3%), followed by Ondarroa (5%) and Cádiz (2.5%). No Anisakis larvae were found in fish from Málaga and Isla Cristina. Three Anisakis genotypes were identified: Anisakis simplex sensu stricto, Anisakis pegreffii and a hybrid genotype between these two species. A. pegreffii was the most prevalent species in A Coruña (71% of larvae). Only three Anisakis larvae (9% collected larvae) were located in the musculature of sardines: two were identified as A. pegreffii while the other was a hybrid genotype. Sardine infection was associated with fishing area and fish length/weight (length and weight were strongly correlated; Pearsons correlation 0.82; p<0.001). Risk factor multivariate analysis showed that the risk of infection increases 1.6 times for every additional cm in the length of the sardines from the same fishing area. Comparison of fish of equal length showed that in sardines from A Coruña the risk of parasitization is 11.5 times higher than in those from other fishing areas. Although the risk of infection by Anisakis through consumption of sardines is generally low due to the low epidemiological parameter values (prevalence 10%, mean intensity 1.7 (range 1-5) and mean abundance 0.17), as larger fish are more heavily parasitized, there is an increased risk of infection by Anisakis through consumption of large sardines which are raw or have undergone insufficient treatment (undercooked, smoked, marinated, salted, pickled, freezing,…).

Activity of key enzymes in glucose catabolism during the growth and metacyclogenesis of Leishmania infantum

Abstract This paper follows the development in the activity of the key enzymes of glycolysis and dehydro- genases of the pentose phosphate shunt throughout the in vitro growth and metacyclogenesis of two human strains of Leishmania infantum– one visceral (VL) and the other cutaneous (CL) – together with changes in the glucose, ammonium, and proton concentrations in the culture medium. In the first stage, ammonium was generated and no glucose was consumed. Later on, all the glucose was consumed and, finally, ammonium was generated again. The ammonium concentration increased 16- and 21-fold in cultures of VL and CL strains, respectively. The activities of the glycosomal enzymes hexokinase and phosphofructokinase differed in each strain, always being higher in CL than in VL and increasing throughout the culture period in CL while decreasing in VL. This probably indicates a different capability to adapt to the culture medium conditions. The activities of the pentose phosphate shunt enzymes examined indicate that 6-phosphogluconate dehydrogenase is possibly a rate-limiting enzyme for this pathway. Pyruvate kinase is a cytosolic control enzyme of glycolysis in trypanosomatids, and its activity decreased throughout the growth and differentiation of both strains of L. infantum, as occurs in other trypanosomatids. It was also observed that glucose catabolism was more active in the cutaneous strain than in the visceral one.

CO2-fixing enzymes during moulting from third larval to fourth larval stage of Anisakis simplex and Hysterothylacium aduncum (Nematoda: Anisakidae)

The fixing of CO2 is an important metabolic process for many organisms. In the anisakid nematodes, CO2 has been shown to be necessary for their development, at least in vitro. The presence of CO2 stimulates the moulting (M3) of the larvae from the third (L3) to the fourth (L4) stage and prolongs the survival, at least, in vitro. We determined the activity of CO2-fixing enzymes, common to many organisms, in two anisakids: Anisakis simplex, a parasite of cetaceans, and Hysterothylacium aduncum, a parasite of fish. Although no activity was detected for pyruvate carboxylase or carboxylating-malic enzyme, we detected phosphoenolpyruvate carboxykinase (PEPCK) and phosphoenolpyruvate carboxylase (PEPC) activity. In A. simplex, PEPCK was clearly higher than that of PEPC throughout the moulting process studied. In H. aduncum, although the activity of both enzymes was of similar magnitude, they showed different behaviour; PEPCK activity decreased after the moulting to L4, PEPC activity increased so that the ratio PEPCK/PEPC activity decreased from 1.90 before moulting to 0.59 after.

Collagenolytic activity related to metalloproteases (and serine proteases) in the fish parasite Hysterothylacium aduncum (Nematoda: Anisakidae).

Proteases play a vital role in both the life cycle of parasites and the parasite-host relationship and are considered important virulence factors. In the present study, the presence of proteases with collagenolytic activity was investigated in the fish nematode Hysterothylacium aduncum during in vitro development. Collagenolytic activity was found in all studied developmental stages of the nematode (third [L3] and fourth [L4] larval stages and adults). In L3, the activity was maximum at pH 6.5 and, in the other stages, at 7.0. Pepsin is known to favour in vitro development of the worm, but, in this study, collagenolytic activity was shown to be significantly greater when no pepsin was added to the culture medium (at pH 6.5, p = 0.011). At pH 7.0, most activity was observed in the immature adult, after the final moult, suggesting that the collagenolytic activity may be involved in remodelling of the cuticle and in sexual maturity. On the other hand, at pH 6.5, activity may be related to tissue migration by L3 within the host. Using specific inhibitors, it was demonstrated that most of the collagenolytic activity detected in all the developmental stages was due to metalloproteases (40 to 100%), although serine proteases were also detected in L4 and adults (10 to 30%).

Helminth infection in Mugil incilis from Cartagena de Indias, Colombian Caribbean coast

SummaryThis survey records the occurrence of larvae of Contracaecum multipapillatum sensu lato (Von Drasche, 1882) Lucker, 1941 (Nematoda, Anisakidae) and adults of Floridosentis mugilis (Machado Filho, 1951) Ward, 1953 (Acanthocephala, Neoechinorhynchidae) in mullet Mugil incilis Hancock, 1830 caught in La Boquilla, Cartagena de Indias (Colombian Caribbean coast). The prevalence of F. mugilis was 45.7 % and of C. multipapillatum s.l. 58.6 %, while 28.6 % of the fish were parasitized by both species. The smallest hosts presented the lowest prevalence of F. mugilis but the highest prevalence of C. multipapillatum s.l.. Prevalence, mean abundance and mean intensity of C. multipapillatum s.l. in M. incilis were all highest in viscera. The prevalence in fish muscle tissue was low (2.9 %).