Anisakid parasites of the pouting (Trisopterus luscus) from the Cantabrian Sea coast, Bay of Biscay, Spain.

An epidemiological survey was undertaken of anisakids in 139 specimens (length: 13.2-24.5 cm) of pouting or bib (Trisopterus luscus) captured off the coast of northern Spain in the Cantabrian Sea. Third-stage larvae of two species of nematodes, Anisakis larvae type I and Hysterothylacium aduncum, were isolated. One adult female H. aduncum was also detected in the intestine of one pouting. Total prevalence of anisakids was 88.5%. Hysterothylacium aduncum and Anisakis showed, respectively, prevalence of 87.8% and 22.3%, mean intensity of 19.7 and 3.5, and mean abundance of 17.3 and 0.8. Analysis of infection parameters as a function of host length revealed a much higher prevalence in pouting specimens with length < 20 cm (94.4% for H. aduncum; 28.0% for Anisakis) than in those with length > or = 20 cm (65.6% for H. aduncum; 3.1% for Anisakis). The high mean intensity of Anisakis in muscle of parasitized pouting (5.9) may pose human health risks, although these are minimized by eating only thoroughly cooked pouting, as is the custom in Spain.

Anisakid parasites of two forkbeards (Phycis blennoides and Phycis phycis) from the Mediterranean coasts of Andalucía (Southern Spain).

An epidemiological survey of anisakids was carried out on 209 specimens of greater forkbeard (Phycis blennoides) and 58 of forkbeard (Phycis phycis) captured off the Mediterranean coasts of Andalucía (southern Spain). Four species of nematodes were identified: Anisakis simplex s.l., Anisakis physeteris, Hysterothylacium aduncum and Hysterothylacium fabri. The total prevalence was 62.06% in the forkbeard and 58.85% in the greater forkbeard. The highest values of prevalence (56.90%), mean intensity (5.21) and mean abundance (2.96) were all obtained for H. fabri in the forkbeard. The most frequent parasite in the greater forkbeard was H. aduncum with 51.20% prevalence and values of 3.00 and 1.53 for mean intensity and mean abundance, respectively. The infestation parameters were also analyzed according to the host length, observing a maximum of parasitized fish (91.67%) in samples of P. blennoides longer than 40 cm, while in P. phycis, the highest prevalence (82.35%), conditioned by H. fabri parasitization, was found in fish with lengths between 30 and 35 cm.

CO(2)-fixing enzymes during moulting from third larval to fourth larval stage of Anisakis simplex and Hysterothylacium aduncum (Nematoda: Anisakidae).

The fixing of CO(2) is an important metabolic process for many organisms. In the anisakid nematodes, CO(2) has been shown to be necessary for their development, at least in vitro. The presence of CO(2) 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 CO(2)-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.

In vitro development of the fish parasite Hysterothylacium aduncum from the third larval stage recovered from a host to the third larval stage hatched from the egg.

Anisakids are parasitic nematodes of fish worldwide, producing economic and human health concerns. It is thus important to ascertain their in vitro life cycle in laboratory studies. Here we describe the in vitro development of third-stage larvae (L3) of Hysterothylacium aduncum isolated from blue whiting Micromesistius poutassou, to the hatching of L3 from eggs obtained from H. aduncum worms grown in GLIT medium (a modified mixture of Yaeger's LIT [Liver Infusion Tryptose] and Grace's media) at pH 4.0, 13 degrees C and with 5% CO2 in air. Under these conditions, L3 recovered from fish developed to mature adults (3.4 to 6.2 cm in length), with oviposition starting from Day 26 in culture. Fertilized eggs (mean size 64 x 52 microm) had a thick, rugose eggshell and were larger than unfertilized ones (mean size 49 x 42 microm), whose eggshells were thin and smooth. Eggs laid during the first and second week of oviposition, and maintained in 2.8% NaCl solution at 13 degrees C, developed to L3. Under these maintenance conditions, between 20.6 and 52.5% of the eggs laid during the first week developed into larvae. Motile larvae, enclosed in a sheath, hatched from between 2 and 11% of these eggs. The larvae started to hatch 23 d after deposition. These larvae were 144 to 215 microm in length, enclosed in a 237 to 305 microm-long sheath. This GLIT culture medium may help to study the biology of this and other anisakids.

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.

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.

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.

Studies on the transmission of Theileria annulata to cattle by the tick Hyalomma lusitanicum.

The role of the ixodid tick Hyalomma lusitanicum Koch 1844 as a vector of Mediterranean or tropical theileriosis (caused by the protozoan parasite Theileria annulata Dschunkowsky et Luhs 1904) in southern Spain was studied. Hyalomma lusitanicum was the most common tick, and the only species of the genus Hyalomma L., found on T. annulata-infected cattle from the theileriosis enzootic area studied (province of Cádiz, southern Spain). Likewise, we found that all sera of the cattle previously considered as suspected of theileriosis by clinical signs, tested for T. annulata antibodies, were positive and all blood samples of these suspected cattle examined had infected erythrocytes. Partially fed H. lusitanicum adults were collected in the field on T. annulata-infected cattle in this enzootic area and fed on an uninfected calf in an experimental farm free of theileriosis and ticks. At approximately 3 weeks post-tick feeding on the calf, this became positive for T. annulata antibodies and T. annulata merozoites were found in erythrocytes from blood smears. These results show the ability of H. lusitanicum to transmit the protozoan parasite T. annulata to susceptible cattle and indicate that H. lusitanicum is probably an important vector of T. annulata in the enzootic area surveyed.

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

This paper follows the development in the activity of the key enzymes of glycolysis and dehydrogenases 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.

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

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.

Investigations on the in vitro metacyclogenesis of a visceral and a cutaneous human strain of Leishmania infantum.

The in vitro metacyclogenesis of a visceral (VL) and cutaneous (CL) human strain of Leishmania infantum was monitored in order to find out the kinetics of this process and the in vitro infective capacity for macrophages of the metacyclic promastigotes developed. To identify, enumerate, and separate the metacyclic population, the complement-dependent lysis by normal serum and the agglutination by peanut agglutinin (PNA) were used, as they were shown to be useful for the purpose of this study. Maximum percentage of metacyclics was detected by both techniques on the 4th day of growth for VL and the 6th day for CL, and was higher for the VL strain. The in vitro infectivity for macrophages of two strains was assayed, and the high parasitization data obtained were transformed in order to determine the increase of the parasite burden for macrophages throughout the incubation time of the experiments (2-72 h post-infection (p.i.)). This parameter is denominated the infectivity ratio (%I) and calculated as follows: (number of intracellular parasites per infected macrophage at 'x' time p.i./number of intracellular parasites per infected macrophage at 2 h p.i.) x 100. When %I was calculated for promastigotes unagglutinated by PNA (PNA-)--metacyclic or infective promastigotes--at any time of culture, the %I at 72 h p.i. was always much higher than for agglutinated promastigotes (2.1-12.5 times)--non-infective promastigotes--and unfractionated promastigotes from culture (1.7-9.5 times), especially with VL parasites. Likewise, the %I for VL PNA- promastigotes from the 4th day of culture was 1.9 times higher than for CL PNA- promastigotes from the 6th day of culture. The higher resistance to lysis by serum, percentage of metacyclics (PNA-), and infectivity ratio of VL than CL could be related to a higher spreading capability into the host body associated with higher pathogenic effects of the visceral strain than the cutaneous one.

Development of an experimental tissue culture vaccine against Mediterranean theileriosis in Spain.

Vaccines against Mediterranean theileriosis have been developed in several countries where the disease is an economic problem. Tissue culture vaccines have been widely and successfully used to immunize cattle. Although Mediterranean theileriosis represents a constraint to dairy cattle production in Spain, no vaccines against this disease have been developed previously. The successful development of a tissue culture vaccine consisting of attenuated Theileria annulata schizont infected cells from an enzootic area of Spain and its efficacy under experimental conditions is reported. Vaccinated calves were resistant to homologous challenge showing no signs of theileriosis while non-vaccinated calves showed typical signs of disease.

Field trial of immunization with an experimental vaccine against Mediterranean theileriosis in Spain.

Vaccines against Mediterranean theileriosis have been developed in several countries where this disease is of economic concern. Until recently, tissue culture vaccines were a suitable method for immunizing cattle and they have been widely applied with success. In Spain, Mediterranean theileriosis is an obstacle to the improvement of dairy cattle productivity. No vaccines against this disease have been applied until recently. This report concerns the field trial of an available experimental tissue culture vaccine consisting of attenuated Theileria annulata schizont infected cells from an enzootic area of Spain. The vaccinated cattle developed a typical post-vaccination immunological response and were resistent to a field challenge. They showed no clinical signs of theileriosis while 50% of the control cattle showed typical signs of the disease and two of them died (12.5% of control cattle). This vaccine may be useful to protect cattle against Mediterranean theileriosis in enzootic areas of Spain.

Some factors which influence the in vitro maintenance of Anisakis simplex (Nematoda).

Several culture media as well as some factors that may affect the in vitro development of the nematode Anisakis simplex Rudolphi, 1809 have been studied. After testing six media and four temperatures, the conditions for the in vitro culture selected were as follows: RPMI-1640 + 20% (v/v) heat-inactived fetal bovine serum or Meyer's M3 (without agar) media, at 37 degrees C, under 5% CO2 in air atmosphere, and renewal of the medium twice a week. The average survival rates of the larvae were significantly increased when the pH of the culture medium was increased (from 4.0 to 7.2) or decreased (from 7.2 to 4.0) after L3 to L4 moulting. The length of the larvae at the onset of culture affected the survival and moulting of themselves, but these were culture medium-dependent. On the other hand, we have observed that several L3 and L4 were attached, by means of a brown unknown substance apparently secreted by themselves, to the bottom of the substratum. Frequently, when a larva was spontaneously detached, a "cap" of the brown substance blocked, apparently, its mouth. The possible absorption of nutrients through the L3 larvae cuticle of A. simplex is discussed.

Isolation and establishment in in vitro culture of a Theileria annulata--infected cell line from Spain.

The isolation of Theileria annulata-infected lymphocytes using blood from an animal suffering from Mediterranean theileriosis as a source of parasites is described. The present work reports the first isolation and establishment in in vitro culture of a T. annulata-infected cell line from southwestern Europe, where Mediterranean theileriosis causes important economic losses, especially in southern Spain. The parasite was identified by staining of cells from culture with Giemsa, by immunofluorescent antibody techniques (IFAT), and by isoenzyme characterization. The possibility of using this T. annulata-infected lymphoblastoid cell line to obtain an antigen for diagnosis of Mediterranean theileriosis by IFAT and to develop a tissue-culture vaccine against this disease in our geographic area shows the significance of this isolation and culture.

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

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

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 South-east 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.

Phloretin and citrate promote the differentiation rate from epimastigote to metacyclic forms of Trypanosoma cruzi.

We have investigated the effects of the metabolic inhibitors, phloretin, 2-deoxy-D-glucose, maleate and trans-aconitate, as well as two intermediates of the tricarboxylic-acid cycle, acetate and citrate, on the growth and metacyclogenesis of Trypanosoma cruzi. 0.1 mM phloretin increased the percentage of metacyclic forms about 2.7-fold without affecting growth rate, whereas the other inhibitors had no apparent effect on either growth or differentiation rates. The addition of 5 mM citrate stimulated differentiation by about 2.6-fold. When either 10 mM citrate or 10 mM acetate were added, on the other hand, both the growth and differentiation rates were severely inhibited.

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. II. Hexokinase, phosphofructokinase and pyruvate kinase.

The 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 microM. Metacyclic forms showed a specific activity of 55.25 mUnits/mg and a Km value of 46.3 microM. 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.