Prevalence of ectoparasitic arthropods on wild animals and cattle in the Las Merindades area (Burgos, Spain).

This paper reports the prevalence of ectoparasitic arthropods in sampled groups of wild (n = 128; 16 species) and domestic (n = 69; 3 species) animals in the Las Merindades area of the Province of Burgos, Spain. The study revealed that wild animals were more infested and with a wider variety of ectoparasites than domestic animals. The parasitic prevalence was 67% for wild animals and 48% for livestock. In this way, 39% of animals were infected by ticks. Ixodes ricinus and Ixodes hexagonus were the most prevalent species whereas Dermacentor reticulatus showed affinity for the fox and wolf. The overall prevalence of parasitisation by fleas was 27%. Ctenophthalmus spp. showed the wider range host in wild animals, while Pulex irritans was the most frequent specie found. The parasitic prevalences by lice (Trichodectes melis, Trichodectes canis and Trichodectes mustelae) and by mite (Neotrombicula spp., Laelaps agilis and Sarcoptes scabiei) were 4% and 12%, respectively. In both cases only wild animals were found parasited.

Some hydrolase activities from the tick Hyalomma lusitanicum koch, 1844 (Ixodoidea: Ixodida).

In this work has been made a detection and preliminary characterization of some hydrolases in whole extracts from unfed adult males and females of Hyalomma lusitanicum, one of the vectors for Theileria annulata that causes Mediterranean theileriosis in cattle. We have elected as targets, proteases as enzymes implicated in the nutritional processes of ticks, esterases that are usually implicated in resistance to organophosphates and phosphatises often implicated in protein phosphorilation and control of ticks salivary gland. The biological role and physiological significance are discussed in terms of the possibility of use these enzymes as possible in future anti-tick vaccination or acaricide resistance.

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.

Trichomonas vaginalis: determination of acid phosphatase activity as a pharmacological screening procedure.

A simple method to screen trichomonacides, based on the quantification of acid phosphatase (AP) activity, has been designed. Using p-nitrophenyl phosphate as chromogenic substrate, we first determined the optimal conditions for enzyme reaction. After seeding, a linear correlation between number of trichomonads and optical densities at 405 nm was obtained at 24 hr but not at 48 hr. Then, the inhibitory effect of metronidazole was assessed both by microscope counts and by AP determination. Similar values for 50% inhibitory concentrations (2.6 microM), with 95% confidence limits of 1.91-3.33 for microscopic and 2.21-3.05 for colorimetric method, were obtained. We concluded that the colorimetric method described in this investigation is suitable for pharmacological studies and for the screening of new, potential antitrichomonal agents.

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.

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)

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.

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.

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.

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.

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 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.

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 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.