Influence of host age and sex on the helminth fauna of the yellow-legged gull (Larus michahellis) in Galicia (northwestern Spain).

We studied the influence of host age and sex on the helminth fauna of 324 Larus michahellis captured in different locations in the region of Galicia (northwestern Spain). Gulls were grouped into prefledglings, first-year immature birds, second- and third-year immature birds, and adults. Second-year, third-year, and adult birds were grouped by sex. Thirty-six helminth species were recorded. Total species richness and mean infracommunity species richness were both significantly lower for pre-fledglings than for the other age groups. Prevalence increased significantly with age for Brachylecithum microtesticulatum, probably reflecting changing feeding habits. Likewise, 8 species (Cardiocephaloides longicollis, Microphallus similis, Maritrema gratiosum, Gynaecotyla longiintestinata, Brachylecithum microtesticulatum, Himasthla elongata, Parorchis acanthus, and Renicola sp.) were absent or had very low prevalence in prefledglings. At least 5 of these 8 species are transmitted to gulls through ingestion of molluscs or crustaceans, which suggests that these types of prey are seldom fed to prefledglings. In Gymnophallus deliciosus, G. longiintestinata, and Cosmocephalus obvelatus, mean intensity, and in the latter case prevalence, declined with age, suggesting that protective immunity against these species increase with age. Only G. deliciosus, Microphallus similis, and G. longiintestinata presented significant differences between the sexes.

Macroparasites of five species of ray (genus Raja) on the northwest coast of Spain.

A parasitological study of rays captured on the Atlantic continental shelf off the estuary Muros-Noia in NW Spain (42 degrees 35' to 42 degrees 41' N, 9 degrees 2' to 9 degrees 10' W; mean capture depth 11.6 +/- 4.1 m) was performed. A total of 128 rays were examined: 52 specimens of Raja microocellata, 60 of R. brachyura, 6 of R. montagui, 3 of R. undulata and 7 of an unidentified Raja species, known locally as 'fancheca'. A total of 23 macroparasite species were detected: 5 monogeneans (Acanthocotyle sp., Calicotyle kroyeri, Empruthotrema raiae, Merizocotyle undulata, Rajonchocotyle emarginata), 11 cestodes (Acanthobothrium sp., Crossobothrium sp., Echeneibothrium sp., Echinobothrium brachysoma, Grillotia erinaceus, Grillotia sp., Lecanicephalum sp., Nybelinia lingualis, Onchobothrium uncinatum, Phyllobothrium lactuca, Tritaphros retzii), 6 nematodes (Anisakis simplex, Hysterothylacium sp., Histodytes microocellatus, Piscicapillaria freemani, Proleptus sp., Pseudanisakis baylisi) and a copepod (Holobomolochus sp.). All parasite species were present in several ray species, except for Acanthocotyle sp. and G. erinaceus (detected only in R. brachyura), H. microocellatus (detected only in R. microocellata) and T. retzii (detected only in R. montagui). Three species (C. kroyeri, M. undulata, E. brachysoma) have not been reported previously from Spain. The host with the highest parasite species richness was R. brachyura (18 species), followed by R. microocellata (17) and the unidentified Raja species (14). The parasite with the highest prevalence in R. microocellata was M. undulata, followed by R. emarginata, Acanthobothrium sp. and Echeneibothrium sp. The species with the highest prevalence in R. brachyura was R. emarginata, followed by C. kroyeri and P. baylisi. Some differences in parasite prevalence were detected between sexes and among size classes in both R. brachyura and R. microocellata.

Ultrastructure and phylogeny of Philasterides dicentrarchi (Ciliophora, Scuticociliatia) from farmed turbot in NW Spain.

Several species of opportunistic histophagous scuticociliates have been implicated in systemic infections of farmed fish. In turbot, scuticociliatosis is an emerging disease, and the identification of the parasite species involved is controversial. We have previously isolated Philasterides dicentrarchi from farmed turbot scuticociliatosis outbreaks in northwest Spain. In the present study, we report detailed ultrastructural studies of this parasite, and investigate phylogenetic relations with other members of the order Philasterida on the basis of sequence comparison of the small-subunit rRNA (SSUrRNA) gene. Ultrastructural study indicates the presence of dikinetids in the anterior two-thirds of the body; micronucleus closely associated with the macronucleus, though not physically connected; numerous mitochondria located below the cell cortex, parallel to the surface; numerous spherical and fusiform extrusomes located close to the plasma membrane. We consider that these characteristics are useful for diagnosis of infections by this parasite. A nested 350-bp nucleotide sequence of the SSUrRNA gene of the turbot P. dicentrachi isolate showed high identity with previously reported SSUrRNA gene sequences from 2 scuticociliates isolated from olive flounder Paralichthys olivaceus in Korea, namely P. dicentrarchi (98%) and Miamiensis avidus (99%); conversely, our P. dicentrarchi sequence showed low identity (86%) with that of Uronema marinum, a scuticociliate that has also been implicated in scuticociliatosis outbreaks in turbot in Europe and olive flounder in Asia. Phylogenetic tree construction on the basis of the SSUrRNA gene sequences, using the neighbour-joining method, confirm that the different P. dicentrarchi isolates and M. avidus are closely related and a possible synonymy between both ciliates species should be considered.

Helminth fauna of the yellow-legged gull Larus cachinnans in Galicia, north-west Spain.

Thirty-six helminth species were found in 324 gulls examined during June 1994 to February 1996 from different localities of Galicia: 25 trematodes (Brachylaima sp., Brachylecithum microtesticulatum, Cardiocephaloides longicollis, Cryptocotyle lingua, Cryptocotyle concavum, Diplostomum spathaceum, Echinostephilla virgula, Galactosomum phalacrocoracis, Gigantobilharzia acotylea, Gymnophallus deliciosus, Gynaecotyla longiintestinata, Himasthla elongata, Himasthla quissetensis, Knipowitschiatrema nicolai, Levinseniella (Levinseniella) propinqua, Maritrema gratiosum, Maritrema linguilla, Microphallus primas, Microphallus similis, Ornithobilharzia canaliculata, Parorchis acanthus, Phagicola minuta, Psilostomum brevicolle, Renicola sp. and Stephanoprora denticulata), four cestodes (Alcataenia micracantha, Microsomacanthus ductilis, Tetrabothrius (Oriana) erostris and Wardium cirrosa), six nematodes (Anisakis simplex, Contracaecum rudolphii, Cosmocephalus obvelatus), Eucoleus contortus, Paracuaria adunca and Tetrameres (Tetrameres) skrjabini) and one acanthocephalan (Arhythmorhynchus longicollis). Tetrabothrius erostris was the most prevalent species (79.6%), followed by C. obvelatus (47.8%), C. lingua (37.4%), G deliciosus (30.9%), G. longiintestinata (22.8%), P. adunca (21.9%), B. microtesticulatum (17.6%), E. contortus (14.5%) and M. similis (9.3%). Microphallus similis was the dominant species, with a Berger-Parker index (BP) of 0.32, followed by T. erostris (BP=0.10). All species presented an aggregated dispersion except G. acotylea and G. phalacrocoracis, which showed a random dispersion. Species that seem to have the greatest predilection for specific sites along the intestine are: C. longicollis and A. micracantha (first third), Brachylaima sp., M. similis and G. longiintestinata (last third) and A. longicollis (second half). Eight species are known to be pathogenic to commercially important fish or molluscan species and several are pathogenic to humans.

Blood protozoans in elasmobranchs of the family Rajidae from Galicia (NW Spain).

Blood smears from 132 skates Raja spp. captured on the continental shelf off Galicia (NW Spain) were examined for blood parasites. The skate species were Raja brachyura (n=60), R. microocellata (n=52) and a total of 20 specimens belonging to R. undulata, R. montagui and another 2 unidentified Raja species, all captured between March 1999 and March 2000. Two blood parasite species were found, Trypanosoma giganteum and Haemogregarina delagei. Of the 132 skates, 16% were infected only by T. giganteum, 17% only by H. delagei, and 5% by both T. giganteum and H. delagei. Both parasites showed highest prevalence in R. brachyura (22% T. giganteum only, 38% H. delagei only, 12% T. giganteum and H. delagei). Mean leucocyte percentages (n=132 fish) were lymphocytes (43%), eosinophils (35%), neutrophils (20%) and monocytes (2%); basophils were not found. As far as we are aware, this eosinophil percentage is the highest reported to date for elasmobranchs. We did not detect any statistically significant differences in leucocyte percentages between infected and uninfected fish, between male and female fish, among species or among weight groups.

Chemotactic responses of the fish-parasitic scuticociliate Philasterides dicentrarchi to blood and blood components of the turbot Scophthalmus maximus, evaluated using a new microplate multiassay.

This study describes a new capillary-type microplate multiassay for characterization of protozoal chemotactic responses, allowing up to 32 assays to be run simultaneously. We used the new multiassay to evaluate the chemoattractant activity of turbot blood components and turbot cells for the facultative parasite Philasterides dicentrarchi, which is responsible for significant losses in turbot farming. Preliminary tests indicated that the assay requires 3-4 h for detection of chemoattractant activity, that it can be performed effectively using the ciliate axenic culture medium, and that it distinguishes clearly between different concentrations of chemoattractant. Application of the assay indicated that whole blood and serum from normal turbot, and especially infected turbot, have strong chemoattractant activity for P. dicentrarchi trophozoites, whereas neither turbot blood cells nor other turbot cells nor bacteria were significant chemoattractants. These results raise the possibility that turbot serum components are involved in host detection and host invasion by P. dicentrarchi, in line with previous findings indicating that turbot with skin lesions show increased susceptibility to P. dicentrarchi infection.

Cysteine proteinase activities in the fish pathogen Philasterides dicentrarchi (Ciliophora: Scuticociliatida).

This study investigated protease activities in a crude extract and in vitro excretion/secretion (E/S) products of Philasterides dicentrarchi, a ciliate fish parasite causing economically significant losses in aquaculture. Gelatin/SDS-PAGE analysis (pH 4, reducing conditions) detected 7 bands with gelatinolytic activity (approximate molecular weights 30-63 kDa) in the crude extract. The banding pattern observed in analysis of E/S products was practically identical, except for 1 low-molecular-weight band detected in the crude extract but not in the E/S products. In assays with synthetic peptide p-nitroanilide substrates, the crude extract hydrolysed substrates characteristic of cysteine proteases, namely Z-Arg-Arg pNA, Bz-Phe-Val-Arg pNA and Z-Phe-Arg pNA. These activities were strongly inhibited by the cysteine protease inhibitor E-64 and by Ac-Leu-Val-Lys aldehyde, a potent inhibitor of cysteine proteases of the cathepsin B protease subfamily. The proteases present in the crude extract degraded both type-I collagen and haemoglobin in vitro, consistent with roles in tissue invasion and nutrition respectively. Again, E-64 completely (collagen) or markedly (haemoglobin) inhibited this degradation. Finally, the histolytic activity of the ciliate in turbot fibroblast monolayers was strongly reduced in the presence of E-64, confirming the importance of secreted cysteine proteinases in the biology of Philasterides dicentrarchi.

In vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate on the scuticociliate fish pathogen Philasterides dicentrarchi.

This study investigated the in vitro effects of the polyphenols resveratrol, mangiferin and (-)-epigallocatechin-3-gallate (EGCG) on the histiophagous ciliate Philasterides dicentrarchi, which causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus L. Of the 3 polyphenols, resveratrol showed strongest antiprotozoal activity, reducing ciliate density after 1 wk culture by, on average, 91% at 50 microM, and 96% at 500 microM. EGCG reduced ciliate density by, on average, 93% at 500 microM, with no significant effect at 50 microM. Mangiferin reduced ciliate density by, on average, 56% at 500 microM, again with no significant effect at 50 microM. In view of these findings, we discuss the potential utility of chemotherapy with polyphenols as a strategy for the control of scuticociliatosis in farmed turbot.

Philasterides dicentrarchi (Ciliophora:Scuticociliatida) expresses surface immobilization antigens that probably induce protective immune responses in turbot.

Philasterides dicentrarchi is a histophagous ciliate causing systemic scuticociliatosis in cultured turbot. This study demonstrates that turbot which survive this disease have serum antibodies that recognize ciliary antigens of this ciliate in ELISA and immobilize/agglutinate the ciliate in vitro. Mouse sera raised against ciliary antigens and integral membrane proteins are likewise capable of immobilizing/agglutinating the ciliates, indicating that P. dicentrarchi, like other ciliates, expresses surface immobilization antigens. Furthermore, the antigen agglutinating reaction induces the parasite to shed its surface antigens rapidly, replacing them with others with different specific serology. This antigen shedding and variation response is similar to that detected in other protozoan parasites. Immunization of turbot with ciliate lysate plus adjuvant or with formalin-fixed ciliates induced synthesis of agglutinating antibodies and conferred a degree of protection against challenge infection, suggesting that the response to surface antigens may play an important role in defence against this pathogen, SDS-PAGE and immunoblotting studies indicated the existence of a predominant polypeptide of about 38 kDa in the ciliary antigen and membrane protein fractions, and this may be the principal surface antigen of P. dicentrarchi.

In vitro growth requirements for the fish pathogen Philasterides dicentrarchi (Ciliophora, Scuticociliatida).

Philasterides dicentrarchi is a scuticociliate causing fatal disease in farmed turbot and sea bass. In view of its high virulence and endoparasitic location, this parasite cannot be effectively controlled by formalin baths, and no systemic chemotherapeutic treatments have yet proved effective; immunoprophylaxis may thus be an attractive alternative approach. Since vaccine development is greatly facilitated by axenic culture of the pathogen, we have developed a simple axenic culture system based on commercially available Leibovitz L-15 medium, supplemented with fetal bovine serum, lipids (lecithin and Tween 80), nucleosides and glucose. After 1 week's culture under optimal conditions (salinity 10 per thousand, pH 7.2, temperature between 18 and 23 degrees C), yields of 1-2 x 10(5)cells/ml were obtained. Even cultures with seeding densities as low as 20 cells/ml were found to produce a good yield of ciliates (about 6 x 10(4)cells/ml) after 11 days of incubation. The ciliates thus obtained were free of contamination by other microorganisms, enabling preparation of pure P. dicentrarchi antigens for vaccine development studies.

Antiprotozoals effective in vitro against the scuticociliate fish pathogen Philasterides dicentrarchi.

The histophagous ciliate Philasterides dicentrarchi causes fatal scuticociliatosis in farmed turbot Scophthalmus maximus and sea bass Dicentrarchus labrax. The present study screened 52 candidate antiprotozoals for activity against this pathogen in vitro. Of these compounds, 14 were effective (i.e. killed all ciliates within a 24 h assay period). In descending order of efficacy (minimum lethal concentration 100 to 0.8 ppm), these were niclosamide, oxyclozanide, bithionol sulfoxide, toltrazuril, N-(2'-hydroxy-5'-chloro-benzoyl) 2-chloro-4-nitroaniline, furaltadone, doxycycline hyclate, formalin, albendazole, carnidazole, pyrimethamine, quinacrine hydrochloride and quinine sulfate. Administration in filtered seawater rather than phosphate-buffered saline inactivated doxycycline hyclate and albendazole, and markedly reduced that of bithionol sulfoxide and toltrazuril, suggesting that these compounds may not be effective in bath administration. In view of these findings, we discuss the potential utility of chemotherapy as a strategy for the control of scuticociliatosis in farmed turbot and sea bass.

Philasterides dicentrarchi (Ciliophora, Scuticociliatida) as the causative agent of scuticociliatosis in farmed turbot Scophthalmus maximus in Galicia (NW Spain).

Two outbreaks of scuticociliatosis affecting farmed turbot Scophthalmus maximus in Galicia are described. Moribund fish showed cutaneous ulcers, darkened skin, swimming behaviour alterations, exophthalmos, and/or abdominal distension as a result of accumulation of ascitic fluid in the body cavity. Ciliates were detected in fresh mounts of practically all organs and tissues, including the blood and ascitic fluid. Histopathological studies revealed severe encephalitis and meningitis (associated with different degrees of softening or liquefaction of the brain), necrosis of the hepatic parenchyme, severe oedema of the intestinal wall, degeneration of muscle fibres, hyperplasia of the branchial epithelium, and/or vascular and perivascular inflammation. In some cases, parasites are surrounded by abundant monocytic and lymphocytic infiltrate. We report the morphological and biometric characteristics of this ciliate, which allow identification as Philasterides dicentrarchi. We discuss possible routes of entry into the host, and environmental factors possibly facilitating infection.

A scanning electron microscope study of the nematode Spirura dentata (Spiruroidea) with notes on the morphometric variations in a Spanish population of this species.

This work provides a detailed morphological description by optic and scanning electron microscopy of the nematode Spirura dentara (Monnig, 1938), a parasite of Herpestes ichneumon in southern and central Spain, together with an analyses of the morphometric variations within this population. Our findings in adult specimens include some important characters not recorded in previous descriptions, and contradict these in a few details: a) the four denticles arising from the inner wall of the oral opening are double, not simple as reported in the original description, b) four simple denticles are present, each located between the corresponding double denticles and the central protuberance of the pseudolabium, c) lateral crests are present running along the first third of the body in the adult, starting a short distance behind the deirids, and d) the pair of small submedian post-cloacal papillae mentioned in the original description has not been observed. The fourth-stage larva is described for the first time. The relative growth rate (i.e., growth rate with respect to body length) of the reproductive zone was higher in females. In adult males, the growth of the left (longer) spicule was positive, whereas that of the right (shorter) spicule was negative.

Intestinal helminths of the red fox (Vulpes vulpes L.) in Galicia (Northwest Spain).

The intestinal helminth fauna of 201 specimens of Vulpes vulpes, captured in Galicia (northwest Spain), was investigated. Eight species were found: Toxocara canis (23% of host specimens), Toxocara cati (0.5%), Toxascaris leonina (1%), Uncinaria stenocephala (28%), Seuratascaris numidica (0.5%), Taenia crassiceps (23%), Mesocestoides litteratus (2.5%) and Dipylidium caninum (0.5%). We discuss possible relationships between human population density and the prevalence of infection of fox by intestinal helminths.

Biovars of coagulase-positive staphylococci isolated from bovine mastitic milk.

Fifty strains of coagulase-positive staphylococci associated with bovine mastitis were biotyped. Of them, 20 were identified as biovar C (cattle & sheep), 17 as biovar B (poultry & swine), 2 as biovar D (hares) and 1 as biovar F (pigeon & fox). Of the remaining strains, 2 were closely related to human biovar A, 2 failed one property to be classified as biovar B, 3 shared properties of biovars B and D and 3 could not be identified. Bovine strains belonging to biovar C formed an heterogeneous group showing differences in crystal violet growth type and production of alpha haemolysin. Only strains associated with biovar A were Tween 80 positive. The highest incidence of lytic reactions amongst all biovars was with phages 42 E (III) and 102 (IV). Antibiotic resistance was most frequently found in biovar C (65%). The 2 strains in biovar D, one classed as biovar C and one as intermediate, were enterotoxigenic (C or D toxins).