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ollowing the intensification of the Mediterranean aquaculture over the years,Sparicotyle(syn.Microcotyle)chrysophrii(van Beneden and Hesse, 1863; Microcotylidae, Monogenea) became one of the most pathogenic ectoparasites for the gilthead seabream (Sparus aurataL.) aquaculture. It has demonstrated a remarkable versatility to expand over its known host range, accomplish an efficient transfer between the wild and farmed sparid populations over wide geographic areas, and establish itself as the most detrimental gilthead seabream pathogen. Even though the incidence ofS. chrysophriiis closely monitored in marine aquaculture system throughout the Mediterranean, there is a general lack of recent and publicly available data on its epidemiology. Being considered as a parasite whose proliferation is expected to increase under global warming conditions, such lack of data additionally humpers the implementation of integrated pest management strategy at national and Mediterranean level. While EU scientific initiatives greatly contributed to our understanding ofS. chrysophriibiology, ecology and sparicotylosis treatment and pathogenesis, there is more ahead to elucidate about the mitigation of the diseases, particularly towards the development of genomic selection breeding programs of parasite-resilient gilthead seabream, omic-based screening of novel drug targets and reversed vaccinology.

Reports on abdominal tumours in koi carp are scarce and most are from the gonads. Their histological diagnosis is challenging due to the occurrence of mixed populations of neoplastic cells and the few availability of cross-reactive antibodies in fish tissues. The present study aims to provide a histopathological characterization of seventeen gonadal tumours, enriched by a wide antibody panel (vimentin, CD117, placental alkaline phosphatase—PLAP, AE1/AE3 cytokeratin, E-cadherin, proliferating cell nuclear antigen—PCNA, müllerian-inhibiting substance—MIS, GATA4 and Inhibin-α) applied on whole and tissue microarray (TMA) sections. Abdominal enlargement was associated with tumours filling 30%–80% of the abdominal cavity; frequently, the gonads had been completely replaced by neoplastic tissue. Twelve cases were characterized as sex cord-stromal tumours (SCSTs), three as germ cell tumours (GCTs), one as mixed germ cell sex cord-stromal tumour (MGCSCST) and one as carcinoma. By immunohistochemistry, PLAP enabled confirmation of GCTs, ovarian carcinoma and the objective identification of a further cell component in 8 out of the 12 SCSTs that were reclassified as mixed tumours. The use of an immunohistochemical panel can help in refining the histological diagnosis, but the morphological diagnosis still represents the main tool for the characterization of these tumours in koi carp.

Red mark syndrome (RMS) is a non-lethal inflammatory skin disorder spreading in farmed adult rainbow trout (Oncorhynchus mykiss) and reported worldwide. The aetiology is still uncertain, but positive correlation was found between Midichloria-like organism and RMS-affected fish. Here, we describe the first cases of RMS in Bosnia and Herzegovina. The outbreaks under study occurred in two intensive farms during the late winter and spring of 2020. Affected fish showed signs of disease ascribable to RMS, confirmed by pathological and molecular examination.

Red mark syndrome (RMS) is a skin disorder affecting rainbow trout (Oncorhynchus mykiss). The present work aimed to correlate the gross skin lesions affecting 46 fish sampled from farms surveyed for RMS with their microscopic features, identifying histological parameters that may be suggestive of disease progression. Skin lesions were grossly included in one of three categories (types I, II and III) according to the progressive degree of severity. Histological parameters and anti‐proliferating cell nuclear antigen (PCNA) tissue immunoreactivity were semi‐quantitatively assessed. In the dermis, PCNA‐positive lymphocytes, fibroblasts and endothelial cells were indicative of active phlogosis. A significant increase in PCNA‐immunoreactive lymphocytes, from gross type I to type III cases, was found only in the hypodermis. The histological parameters significantly associated with the gross lesion severity were progressive loss of the epithelium and scales, recruitment of inflammatory cells in the stratum compactum, loss of architecture of the stratum compactum, perivascular and perineural granulomatous inflammation and increase in lymphocyte infiltration of the muscular layer. In the type II and type III categories, inflammation in the hypodermis and muscle displayed a granulomatous pattern, reinforcing the hypothesis of an immunopathological mechanism. The morphological diagnosis of “deep chronic dermatitis associated to panniculitis and myositis, characterised by lympho‐histiocytic and granulomatous reaction” is suggested.

Leptospirosis is a worldwide-spread zoonosis causing disease and death in dogs and in humans. A Leptospiral infection has been recorded in several wild carnivore species in Europe, but tissue pathological changes were not commonly described. The Grey wolf (Canis lupus) has been expanding its distribution range in north-eastern Italy during the last decade. A young wolf, representing the first individual handled in the region, was found road-killed and then submitted to necropsy. Pathological changes included erosive lesions of gingival mucosa, mild liver enlargement, and multifocal degenerative-necrotic areas along with hyperemic reactive lesions; multifocal interstitial nephritis and multifocal lung hemorrhages were observed. A Polymerase Chain Reaction (PCR) able to detect pathogenic species of Leptospira performed on a kidney sample was positive. Serological reactions for serogroup Gryppotyphosa (1:6400), Pomona (1:800), and Icterohaemorrhagiae (1:200) were evidenced by MAT. Genotyping by Multilocus Sequence Typing (MLST) performed on detected Leptospira characterized it as belonging to Sequence Type (ST) 117, which refers to L. kirschneri, serogroup Pomona, serovar Mozdok. Regardless of the role of Leptospira infection as an eventual predisposing factor to the road killing of this wolf, to the best of the authors’ knowledge, this is the first report of Leptospira-induced pathology in a wolf in Europe. Surveys on Leptospira infection in free-ranging wildlife species should be pursued in order to achieve further epidemiological knowledge on the circulation of the Leptospira strain.

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the etiological agent of amyloodiniosis in European seabass (Dicentrarchus labrax) (ESB). There is a lack of information about basic molecular data on AO biology and its interaction with the host. Therefore, de novo transcriptome sequencing of AO tomonts was performed. AO trophonts were detached from infested ESB gills, and quickly becoming early tomonts were purified by Percoll&reg, density gradient. Tomont total RNA was processed and quality was assessed immediately. cDNA libraries were constructed using TruSeq&reg, Stranded mRNA kit and sequenced using Illumina sequencer. CLC assembly was used to generate the Transcriptome assembly of AO tomonts. Out of 48,188 contigs, 56.12% belong to dinophyceae wherein Symbiodinium microadriaticum had 94.61% similarity among dinophyceae. Functional annotations of contigs indicated that 12,677 had associated GO term, 9005 with KEGG term. The contigs belonging to dinophyceae resulted in the detection of several peptidases. A BLAST search for known virulent factors from the virulence database resulted in hits to Rab proteins, AP120, Ribosomal phosphoprotein, Heat-shock protein70, Casein kinases, Plasmepsin IV, and Brucipain. Hsp70 and casein kinase II alpha were characterized in-silico. Altogether, these results provide a reference database in understanding AO molecular biology, aiding to the development of novel diagnostics and future vaccines.

Flathead grey mullet, Mugil cephalus, with mass whitish cystlike plasmodia on their scales were collected at Kerchensky preglass of the Black Sea in 2015. The prevalence of infected fish varied from 15% in spring, reached 100% in summer, and declined to 2.5% in autumn. No fish mortality was detected. The spores were oval in frontal view, tapering to a blunt apex. Two unequal polar capsules were pyriform and extended over the anterior half of spore. Spores were 8.2±0.03 µm (7.9-8.4) long, 5.9±0.23 µm (5.2-7.3) wide, and 4.4±0.17 µm (4.0-4.7) thick. Two pyriform and unequal polar capsules were observed (4.0±0.07 µm (3.3-4.5) long, and 1.5±0.24 µm (1.1-1.8) wide). The investigation of nucleotide sequences of the 18S rDNA gene of the myxosporean spores from scales with universal primer А (5′-ACCTGGTTGATCCTGCCAGT-3′) and В (5′-TGATCCTTCTGCAGGTTCACCTAC-3′) showed 100% identity with episquamalis, and 99 % with M. bizerti, M. ichkeulensis, M. spinacurvata and Myxobolus sp. previously detected in mullets. The results obtained from the present study reveal that M. episquamalis, found on the scales of M. cephalus from Kerchensky preglass of the Black Sea, is new record for Russian waters.

The fish parasites Saprolegnia spp. (Oomycota) and Amyloodinium ocellatum (Dinophyceae) cause important losses in freshwater and marine aquaculture industry, respectively. The possible adverse effects of compounds used to control these parasites in aquaculture resulted in increased interest on the search for natural products with antiparasitic activity. In this work, eighteen plant-derived compounds (2′,4′-Dihydroxychalcone; 7-Hydroxyflavone; Artemisinin; Camphor (1R); Diallyl sulfide; Esculetin; Eucalyptol; Garlicin 80%; Harmalol hydrochloride dihydrate; Palmatine chloride; Piperine; Plumbagin; Resveratrol; Rosmarinic acid; Sclareolide; Tomatine, Umbelliferone, and Usnic Acid) have been tested in vitro. Sixteen of these were used to determine their effects on the gill cell line G1B (ATCC®CRL-2536™) and on the motility of viable dinospores of Amyloodinium ocellatum, and thirteen were screened for inhibitory activity against Saprolegnia spp. The cytotoxicity results on G1B cells determined that only two compounds (2′,4′-Dihydroxychalcone and Tomatine) exhibited dose-dependent toxic effects. The highest surveyed concentrations (0.1 and 0.01 mM) reduced cell viability by 80%. Upon lowering the compound concentration the percentage of dead cells was lower than 20%. The same two compounds revealed to be potential antiparasitics by reducing in a dose-dependent manner the motility of A. ocellatum dinospores up to 100%. With respect to Saprolegnia, a Minimum Inhibitory Concentration was found for Tomatine (0.1 mM), Piperine and Plumbagin (0.25 mM), while 2′,4′-Dihydroxychalcone considerably slowed down mycelial growth for 24 h at a concentration of 0.1 mM. Therefore, this research allowed to identify two compounds, Tomatine and 2′,4′-Dihydroxychalcone, effective against both parasites. These compounds could represent promising candidates for the treatment of amyloodiniosis and saprolegniosis in aquaculture. Nevertheless, further in vitro and in vivo tests are required in order to determine concentrations that are effective against the considered pathogens but at the same time safe for hosts, environment and consumers.

not available.

The ectoparasite protozoan Amyloodinium ocellatum (AO) is the causative agent of amyloodiniosis in European seabass (ESB, Dicentrarchus labrax). There is a lack of information about basic molecular immune response mechanisms of ESB during AO infestation. Therefore, to compare gene expression between experimental AO-infested ESB tissues and uninfested ESB tissues (gills and head kidney) RNA-seq was adopted. The RNA-seq revealed multiple differentially expressed genes (DEG), namely 679 upregulated genes and 360 downregulated genes in the gills, and 206 upregulated genes and 170 downregulated genes in head kidney. In gills, genes related to the immune system (perforin, CC1) and protein binding were upregulated. Several genes involved in IFN related pathways were upregulated in the head kidney. Subsequently, to validate the DEG from amyloodiniosis, 26 ESB (mean weight 14 g) per tank in triplicate were bath challenged for 2 h with AO (3.5 × 106/tank; 70 dinospores/mL) under controlled conditions (26−28 °C and 34‰ salinity). As a control group (non-infested), 26 ESB per tank in triplicate were also used. Changes in the expression of innate immune genes in gills and head kidney at 2, 3, 5, 7 and 23 dpi were analysed using real-time PCR. The results indicated that the expression of cytokines (CC1, IL-8) and antimicrobial peptide (Hep) were strongly stimulated and reached a peak at 5 dpi in the early infestation stage, followed by a gradual reduction in the recovery stage (23 dpi). Noticeably, the immunoglobulin (IgM) expression was higher at 23 dpi compared to 7 dpi. Furthermore, in-situ hybridization showed positive signals of CC1 mRNA in AO infested gills compared to the control group. Altogether, chemokines were involved in the immune process under AO infestation and this evidence allows a better understanding of the immune response in European seabass during amyloodiniosis.

Cytauxzoonosis is an emerging, tick-transmitted, protozoan disease affecting domestic and wild felids and caused by Cytauxzoon felis, Cytauxzoon manul and Cytauxzoon spp. This study aimed to determine the presence of infection with Cytauxzoon spp. in Felis silvestris silvestris in Italy, in order to enhance the comprehension of its pattern distribution among domestic cat populations. In addition, wildcats were tested for other endemic vector-borne pathogens in Italy. The carcasses of 21 F. s. silvestris were collected from central and northern regions of Italy. All the animals were submitted to necropsy and samples of the spleens were collected. Cytauxzoon infection was surveyed by a conventional PCR amplifying a portion of the SSU-rDNA of species of Piroplasmida. The samples were also screened for Anaplasma spp., Ehrlichia spp., Rickettsia spp., Babesia spp., Theileria spp., and Leishmania spp. using SYBR Green Real-Time PCR (rPCR) assays. Four animals (19%) were positive for Piroplasmida-PCR assay and three sequenced amplicons were obtained (14.3%), clustering with the Italian, Spanish, French and Romanian Cytauxzoon spp. isolates and with C. manul found in Mongolia. The samples were negative for the other pathogens screened. The present results showed that Cytauxzoon spp. may infect both F. s. silvestris and F. s. catus.

The objective was to examine the potential use of resistant starch (RS) as a fibre-enriching ingredient in gluten-free pasta. Pasta was enriched with commercial RS type II (Hi-Maize™ 260) at 100-200 g kg-1 substitution of rice flour. The effects on the rheological properties of dough and pasta quality as a result of RS addition and the loss in RS due to the process were evaluated.; Results: Dough water absorption was not influenced by the addition of RS. The cooking loss (CL) of RS-enriched samples was 30% lower than reference without fibre. The addition of RS significantly increased firmness of cooked pasta, and above 100 g kg-1 RS level of substitution samples showed a lower stickiness value. Dynamic rheological tests on pasta dough showed a higher storage modulus for fibre samples, indicating a higher number of elastically physical interactions. Loss in RS in uncooked pasta was about 31% compared with the initial amount added to the product.; Conclusions: The addition of RS improved the quality of gluten-free pasta owing to its ability to increase the firmness and decrease the CL and stickiness of cooked pasta. The product enriched with 200 g kg-1 RS can be considered a source of DF. © 2016 Society of Chemical Industry.; © 2016 Society of Chemical Industry.

Extra-intestinal nematodes of companion animals are of growing concern in veterinary medicine for their pathogenic potential and the current expansion throughout Europe. The present study has evaluated the occurrence of major canine and feline extra-intestinal nematodes in regions of Italy having epidemiological relevance. Associations of various recorded parameters related to the examined animals have been statistically evaluated, along with a comparative analysis with the most recent epidemiological data. Overall, 1055 dogs and 1000 cats were tested. Among extra-intestinal nematodes Angiostrongylus vasorum was the most common in dogs followed by Capillaria aerophila and Dirofilaria spp.; Aelurostrongylus abstrusus was the most recorded parasite in cats, followed by C. aerophila and Troglostrongylus brevior. The statistical analysis revealed that outdoor access is associated with A. vasorum, A. abstrusus and T. brevior infections, that were also more prevalent in animals with cardio-respiratory signs. Moreover, cats aged less than 12 months had more chances to be infected by lungworms. The data herein presented confirm the occurrence and the possible risk of expansion of different extra-intestinal parasitoses of dogs and cats in Italy, underlining the importance of a constant epidemiologic vigilance and of appropriate control methods.

In the Mediterranean area, amyloodiniosis represents a major hindrance for marine aquaculture, causing high mortalities in lagoon-type based rearing sites during warm seasons. Amyloodinium ocellatum (AO) is the most common and important dinoflagellate parasitizing fish, and is one of the few fish parasites that can infest several fish species living within its ecological range. In the present study, A. ocellatum was recorded and collected from infected European sea bass (Dicentrarchus labrax) during a summer 2017 outbreak in north east Italy. Histological observation of infected ESB gill samples emphasized the presence of round or pear-shaped trophonts anchored to the oro-pharingeal cavity. Molecular analysis for small subunit (SSU) rDNA of A. ocellatum from gill genomic DNA amplified consistently and yielded 248 bp specific amplicon of A. ocellatum, that was also confirmed using sequencing and NCBI Blast analysis. Histological sections of ESB gill samples were addressed to immunohistochemical procedure for the labelling of ESB igm, inos, tlr2, tlr4, pcna and cytokeratin. Infected gills resulted positive for igm, inos, pcna and cytokeratin but negative to tlr-2 and tlr-4. Furthermore, ESB immune related gene response (innate immunity, adaptive immunity, and stress) in the course of A. ocellatum infection using quantitative polymerase chain reaction (qpcr) for infected gills and head kidney was analysed. Among the twenty three immune related gene molecules tested, cc1, il-8, il-10, hep, cox-2, cla, cat, casp9, and igt were significantly expressed in diseased fish. Altogether, these data on parasite identification and expression of host immune-related genes will allow for a better understanding of immune response in European sea bass against A. ocellatum and could promote the development of effective control measures.

Grapevines store non-structural carbohydrates (NSC) during late summer to sustain plant development at the onset of the following spring’s growth. Starch is the main stored carbohydrate, found in the wood-ray parenchyma of roots and canes. Although the relationship between hydraulic and plant photosynthetic performance is well-recognized, little research has been done on the long-term effects of drought in grapevines adopting different strategies to cope with water stress (i.e. isohydric and anisohydric). We performed our study by exposing two different grape cultivars (Syrah and Cabernet Sauvignon) to a short but severe drought stress, at two stages of the growing season (July and September). No marked differences in the physiological and hydraulic responses of the two varieties were found, probably due to our experimental conditions. However, anatomical and biochemical characterization of overwintering canes pointed out several interesting outcomes. We found a significant and parallel increase of starch and medullar ray number in both cultivars exposed to early water stress. We hypothesize that stressed vines limited their carbon allocation to growth, while shifting it to starch accumulation, with a most evident effect in the period of intense photosynthetic activity. We also speculate that a different aptitude to osmotic adjustment may underlay variation in starch increase and the specific involvement of bark NSC in the two cultivars.

The aim of this study was to investigate the effects of replacing graded levels of dietary fish meal by a blend of two marine microalgae Tisochrysis lutea and Tetraselmis suecica on intestinal morpho-physiology and innate immune response in European sea bass. Two complete diets were formulated to be iso-nitrogenous and isolipidic and prepared by including a blend of the two microalgae, to replace approximately 15 and 45% fish meal protein of the control diet. A fourth diet, where the microalgae mix was substituted by soybean meal, was also prepared. Each diet was offered until visual satiety over 105 days to triplicated groups of European sea bass (204 ± 12.7 g), kept in a recirculating marine water system. The humoral and cellular innate immune parameters of E. sea bass were affected by the dietary treatment. Fish fed the microalgae-containing or the soybean rich diets, showed a significantly greater villi height, while the thickness of intestinal epithelium was significantly reduced in fish fed the soybean meal-rich diet. The activity of the brush border membrane enzymes, maltase, sucrase-isomaltase, γ-glutamil transferase and alkaline phosphatase was not affected by dietary treatment but changed in different intestinal tracts. The genes sucrase-isomaltase, peptide transporter 1, sodium/potassium-transporting ATPase and aminopeptidase N were overexpressed in the pyloric and proximal region of the intestine of fish fed the microalgae-including diets. In conclusion, a blend of dried marine microalgae Tisochrysis lutea and Tetraselmis suecica as alternative ingredients to dietary fish meal did not hamper gut digestive-absorptive functions of E. sea bass. Moreover, it resulted in enhanced non-specific immune response, suggesting an effective role as an immunostimulant ingredient.

Enhanced shrub growth and expansion are widespread responses to climate warming in many arctic and alpine ecosystems. Warmer temperatures and shrub expansion could cause major changes in plant community structure, affecting both species composition and diversity. To improve our understanding of the ongoing changes in plant communities in alpine tundra, we studied interrelations among climate, shrub growth, shrub cover and plant diversity, using an elevation gradient as a proxy for climate conditions. Specifically, we analyzed growth of bilberry (Vaccinium myrtillus L.) and its associated plant communities along an elevation gradient of ca. 600 vertical meters in the eastern European Alps. We assessed the ramet age, ring width and shoot length of V. myrtillus, and the shrub cover and plant diversity of the community. At higher elevation, ramets of V. myrtillus were younger, with shorter shoots and narrower growth rings. Shoot length was positively related to shrub cover, but shrub cover did not show a direct relationship with elevation. A greater shrub cover had a negative effect on species richness, also affecting species composition (beta-diversity), but these variables were not influenced by elevation. Our findings suggest that changes in plant diversity are driven directly by shrub cover and only indirectly by climate, here represented by changes in elevation.

Conventional methods to preserve adult nematodes for taxonomic purposes involve the use of fixative or clearing solutions (alcohol, formaldehyde, AFA and lactophenol), which cause morphological alterations and are toxic. The aim of this study is to propose an alternative method based on glycerol-cryopreservation of nematodes for their subsequent identification. Adults of trichostrongylid nematodes from the abomasum of roe deer (Capreolus capreolus Linnaeus) were glycerol-cryopreserved and compared with those fixed in formaldehyde, fresh and frozen without cryoprotectans. Morphology, transparency and elasticity of the anterior and posterior portion of male nematodes were compared, especially the caudal cuticular bursa and genital accessories. The method presented is quick and easy to use, and the quality of nematode specimens is better than that of nematodes fixed by previously used fixatives. Moreover, glycerol cryopreserved nematodes can be stored for a long time at -20 degrees C in perfect condition and they could be suitable for further analyses, such as histological or ultrastructural examinations.

The influence of ultrasound treatments of tomato pulp on microstructure and lycopene in vitro bioaccessibility was investigated. To this purpose, samples were subjected to ultrasound at a frequency and amplitude of 24 kHz and 100 μm, respectively, for increasing lengths of time. Results showed that ultrasound was responsible for loss of tomato cell integrity, as well as a decrease in the degree of pectin esterification. In contrast, rheological measurements showed that ultrasonically treated tomato pulp had greater gel-like properties than an untreated sample. It was inferred that ultrasound promoted the formation of a new network due to hydrogen bonding and hydrophobic interactions among the de-esterified pectin molecules. Such a reinforcement of the tomato pulp structure resulted in a decrease in lycopene in vitro bioaccessibility of the ultrasonically treated tomato pulp, probably due to the fact that the presence of a stronger network may make lycopene less available to the digestion process.

Red mark syndrome (RMS) and US strawberry disease (US SD) are skin disorders affecting rainbow trout farmed in Europe and USA. The disease etiology has not yet been established. In spite of specific investigations, identifying Rickettsia-like organism (RLO)- and Midichloria-like organism (MLO)-related DNA in affected individuals, these pathogens have never been observed. We performed histological, ultrastructural and biomolecular analysis on skin and spleen samples of trout with RMS. Examination by TEM revealed the presence of intracytoplasmic microorganisms resembling Rickettsiales within macrophages, fibroblasts and erythrocytes. The microorganisms were oval or short rod shaped (400–800 nm in length and 100–200 nm in width) and often showed a cell wall similar to Gram-negative bacteria. PCR analysis for Rickettsiales supported these findings: 53% of affected trout were positive by both PCR and TEM The primers RiFCfw-RiFCrev were used to anneal both the RLO 16S DNA sequence and the MLO 16S DNA sequence. For this reason, and in agreement with previous studies confirming the presence of Rickettsiales-related DNA in trout with RMS, we assume that TEM detected microorganisms morphologically consistent with bacteria belonging to Rickettsiales order and could be considered as possible causative agents of RMS.

Opercular anomalies are very frequent in reared gilthead sea bream and these can negatively influence the product value. Field observations have suggested that opercular malformations can recover over time. In order to verify this hypothesis, 140-day-old gilthead sea bream with monolateral opercular anomalies were divided into three groups, according to the type and increasing seriousness of the opercular malformations, and another group was composed of fish with bilateral opercular anomalies. All groups were monitored for 16 months. In the group with monolateral anomalies, the opercular recovery process was documented by morphological (stereomicroscope) and morphometric analysis. For the latter analysis, two relevant areas, A and T, were identified in the cephalic region. The ratio (T - A)/T, tending to 1, represents the recovery index (RI) of anatomical integrity and quantifies the recovery level of opercular complex anomalies. Results suggested that the recovery process was considerable over the 16 months of investigation but should not be considered complete. At the end of the study, 61% of the gilthead sea bream population with monolateral opercular defects recovered external integrity, whereas the population with bilateral defects showed a poorer recovery capability.

A broad scale study was carried out in 2008 to evaluate the distribution and species-specific occurrence of cyathostomin populations in horse yards from Europe. In total 102 properties and 3123 horses were included in Italy (60 yards and 1646 animals), United Kingdom (22 yards and 737 animals) and Germany (20 yards and 740 animals). Individual faecal samples were examined with a McMaster technique while pooled samples were subjected to the microscopic examination of in vitro cultured larvae and to a Reverse Line Blot (RLB) assay able to molecularly identify the most diffused 13 species of cyathostomins. All yards were positive for the presence of cyathostomins both at the McMaster technique and at the microscopic examination of cultured larvae. One thousand and nine hundred thirty-one horses (61.8%) showed a positive faecal egg count, i.e. 1110 (67.4%), 463 (62.8%) and 358 (48.3%) from Italy, UK and Germany respectively. Out of the 1931 positive animals 1133 (36.3%) showed a faecal egg count per gram >150, specifically 694 (42.2%) from Italy, 237 (32.2%) from UK and 202 (27.3%) from Germany. The molecular results showed that all 13 species that can be detected by the RLB were found in each of the three countries, with a range of 3-13 species present in individual yards. The five most prevalent were Cylicocyclus nassatus, Cylicostephanus longibursatus, Cyathostomum catinatum, Cylicocyclus goldi and Cyathostomum pateratum. The relevance of these results and related biological and epidemiological features are discussed, together with their significance for both future studies of cyathostomins and further intervention programs aiming to control the spread of anthelmintic-resistant populations.

The authors studied a population of 582 gilthead sea breams (Sparus aurata), aged 25–600 days, in order to describe the abnormalities of the opercular complex. Particular attention was paid to: (1) the different degrees of abnormality of the opercular complex as a whole and of the individual dermal bones (preopercle, interopercle, subopercle and opercle) that compose it; (2) the ways in which the operculum folds into the gill cavity and the relevant histological characteristics. This study discusses the origin and nature of the abnormalities. Though these abnormalities have a major impact on market value and product image, and are often linked to other disorders in fish, they are still difficult to eliminate. Gaining a basic understanding of these abnormalities will be a first step towards reducing their occurrence. D 2003 Elsevier Science B.V. All rights reserved.

Summary This report describes a spontaneously arising rhabdomyosarcoma of soft tissues in a brook trout (Salvelinus fontinalis). The lesion was examined by means of histology, immunohistochemistry (IHC) and transmission electron microscopy (TEM). The cross-reactivity of the primary antibodies used in the IHC was investigated in silico using the Protein Blast system. Microscopically, the lesion appeared as a ‘small round cell’ undifferentiated sarcoma with rare myotube formation. IHC identified expression of sarcomeric actin and vimentin and these molecules showed the highest protein sequence identity. Lower protein sequence identity coincided with negative immunolabelling for desmin, MyoD1, myogenin and CD3. TEM revealed myofibrils, but without a defined sarcomeric architecture. The diagnosis of solid alveolar rhabdomyosarcoma of soft tissues was achieved on the basis of histological and ultrastructural findings.

Intestinal parasites are common in dogs worldwide, and their importance has recently increased for a renewed awareness on the public health relevance that some of them have. In this study, the prevalence of helminths and protozoa was evaluated by microscopy in 318 canine faecal samples collected from eight rescue shelters in the North-eastern Italy; 285 of them were also submitted to the molecular characterization of Giardia duodenalis and Cryptosporidium spp. isolates. An analysis was performed to evaluate the prevalence rates in relation to canine individual data, shelter provenance and anthelmintic treatments. Overall, 52.5 % (167/318) of faecal samples were positive for at least one parasite. Trichuris vulpis showed the highest overall prevalence rate (29.2 %), followed by G. duodenalis (15.1 %), Toxocara canis (9.7 %), ancylostomatids (8.2 %) and Cystoisospora (5.7 %). The prevalence of G. duodenalis, evaluated by real-time PCR, was 57.9 % (165/285), and 79 isolates were characterized by nested PCR on the β-giardin gene. The assemblages found were mainly the host-specific genotypes C and D, while only one assemblage was identified as the human-specific genotype B1. Isolates of Cryptosporidium spp., recorded in 3/285 (1.1 %) stool samples, were Cryptosporidium parvum based on the characterization of the Cryptosporidium oocyst wall protein (COWP) gene. Although the results describe a relatively limited risk of dog-originating zoonoses, there is the need to improve the quality of shelter practices towards better health managements for safe pet-adoption campaigns and a minimization of the environmental faecal pollution with canine intestinal parasites.

The occurrence of common respiratory parasites of domestic cats (the metastrongyloid “cat lungworm” Aelurostrongylus abstrusus and the trichuroid Capillaria aerophila) and of neglected respiratory nematodes of felids (Troglostrongylus brevior, Angiostrongylus chabaudi and Oslerus rostratus) was here evaluated in two and three geographical sites of Northern and Central Italy, respectively. In 2014–2015, individual fecal samples of 868 domestic cats were examined microscopically and genetically, and epidemiological data related to parasitic infections were evaluated as possible risk factors by binary logistic regression models. The most common parasite was A. abstrusus in both mono- and poli-specific infections, followed by T. brevior and C. aerophila, while cats scored negative for other parasites. Cats positive for A. abstrusus (1.9–17 % infection rate) and C. aerophila (0.9–4.8 % infection rate) were found in all examined sites, while cats scored positive for T. brevior (1–14.3 % infection rate) in four sites. Also, T. brevior was here found for the first time in a domestic cat from a mountainous area of Northern Italy. The occurrence of lungworms was statistically related to the presence of respiratory signs and more significant in cats with mixed infection by other lungworms and/or intestinal parasites. Cats living in site C of Central Italy resulted statistically more at risk of infection for lungworms than cats living in the other study sites, while animals ageing less than 1 year were at more risk for troglostrongylosis. Finally, the presence of lungworms was more significant in cats with mixed infection by other lungworms and/or intestinal parasites. These results are discussed under epidemiological and clinical points of views.

The high prevalence of opercular deformities present in fish from Mediterranean marine hatcheries is an important problem, which is causing considerable economic losses. The aetiology of this syndrome is not yet well understood. In this study a histological and ultrastructural description of gilthead sea bream (Sparus aurata) larvae affected with anomalies of the opercular complex was carried out.

The dentex ( Dentex dentex L.) is a fast-growing sparid which represents a possible candidate for Mediterranean mariculture. As the basic nutrient requirements of this species are unknown, two feeding trials were carried out to identify suitable protein and lipid (energy) levels to be used in formulating practical diets for dentex. The experiments used groups of 30 specimens (each fish weighing 17 or 20.7 g) which were kept in 12 flow-through 160 1 tanks supplied with sea water at 2 1 min −1 (temperature 20 ± 1 °C; salinity 33 ppt). An initial 4-week trial was conducted to obtain a preliminary estimate of the optimal protein level. Triplicate groups were fed four practical diets with increasing levels of protein (44.3, 49.3, 55.7 and 58.9% dry matter (DM), over 90% of which was supplied by white fish meal) and a constant lipid content (17.3% DM; mostly supplied by fish lipids) to satiation. An analysis of the nutrient-weight gain relationship indicated that the optimal dietary protein level was 49.3% DM. In a second 60 day experiment, duplicate groups of fish were fed six diets with three protein levels (44.4, 49.5 and 55.8% DM) and two lipid contents (12.0 and 17.3% DM) within each protein level, nearly to satiation. Growth performance was lowest in fish given diets containing 44.4 or 49.5% protein and 12% lipid and was improved ( P −1 calculated available energy) could be assumed as suitable levels for formulating practical diets for this species, providing high quality fish meal and oil are used.

The potential exploitation of radiofrequency (RF) to assist food freezing was studied. To this aim, freezing of pork meat was performed in a RF pilot equipment modified to allow food immersion in a liquid nitrogen spray. During freezing, pulsed RF treatments were applied (RF assisted cryogenic freezing). Freezing under cryogenic fluid flow (cryogenic freezing) and air freezing were also performed as controls. Results showed that RF assisted freezing is possible by using low voltage pulses (2 kV). The thawing losses of RF cryogenic frozen meat were much lower than that observed during thawing of air and cryogenic frozen meat, which showed similar drip loss. The analyses carried out on the microstructure of meat showed that the tissue exhibited a better cellular structure when RF was applied. Much less intercellular voids and cell disruption were observed. As compared to the control sample, RF frozen meat presented smaller ice crystals, which were mainly located at intracellular level. This was attributed to the ability of RF to depress the freezing point thus producing more nucleation sites.

Background For the control of worm infections, the strategic use of anthelmintics, often accompanied by additional farm and/or pasture management procedures, is currently applied on most horse farms in industrialized countries. However, the particular effects of the specific worm control procedures are often unclear and have only been investigated to a limited extent. We examined faecal egg count (FEC), faecal egg count reduction (FECR) and questionnaire data on farm and pasture management procedures. The aim of this study was to determine whether specific worm control practices reported to be applied in European horse farms affect worm prevalence. Results This study involved 20 German, 26 Italian and 16 UK horse farms for each of which FEC were performed on a minimum of 16 horses. In total, 2029 horse faecal samples were quantitatively analysed for helminth eggs, resulting in 56.3% of the faecal samples being positive for strongylid eggs. The prevalence in the 742 German horse samples (48.1%) was significantly lower than that in the 737 Italian (61.1%) and the 550 UK (60.9%) samples. As expected, a significant effect of horse age on the infection prevalence was observed, with adult horses showing lower prevalences and lower mean FEC than foals and yearlings. The majority of the participating farms were stud farms (n = 29), followed by riding stables (n = 27) and racehorse stables (n = 6). The prevalence of strongyle infection by farm type differed between countries. While in Germany, horses on riding farms were significantly less often strongyle positive, in the UK horses on stud farms showed the lowest strongyle prevalences, whereas in Italy, no significant difference between farm types were seen. On all farms, horses received routine/preventive anthelmintic treatment. An effect of treatment frequency on strongyle prevalence was only encountered with adult horses. On farms performing more than one annual treatment, faecal samples were significantly less often positive. Furthermore, by comparing the FECR results of individual horses with their pre-treatment FEC, it was found that high pre-treatment FEC were associated with a significantly higher probability for a FECR below 90%. Conclusion Overall, age-dependent strongyle infection patterns and general worm control approaches were found to be similar on horse farms in the three countries. Also, a negative association of pre-treatment FEC and treatment efficacy was consistently found in all countries. However, mean strongyle prevalences and frequencies of anthelmintic treatments were considerably different. In addition to the age-dependent prevalence patterns, the finding of a possible negative association between high FEC and reduced FECR might argue for a focus on horses showing high pre-treatment FEC when monitoring anthelmintic treatment efficacy in the field.

Background A large survey was carried out in 2008 in Europe to evaluate the efficacy of fenbendazole (FBZ), pyrantel (PYR), ivermectin (IVM) and moxidectin (MOX), i.e. the major anthelmintic molecules used in current practice against cyathostomins affecting horses. A total of 102 yards and 1704 horses was studied in three countries: 60 yards and 988 horses from Italy, 22 and 396 from the UK, 20 and 320 from Germany. The survey consisted of Faecal Egg Count Reduction Tests (FECRTs) with a faecal egg count reduction (FECR) categorization of (I) resistance present if FECR 90%. The calculation of FECR data was performed employing bootstrap analysis of group arithmetic means. Results The testing of FBZ on a total of 80 yards resulted in resistance present on more than 80% of the UK and German yards and on significantly fewer in Italy, i.e. in 38% (p < 0.01). PYR, IVM and MOX were tested on a total of 102 yards. For PYR resistance present was found in 25% of the yards with no significant differences between countries. For IVM resistance present was encountered in one Italian and two UK yards (3%), resistance present to MOX was not found in any yard in any country. Conclusion The results indicate that single and/or multiple drug resistance in equine cyathostomins is present in the three countries, is widespread particularly for FBZ and/or PYR and in one UK yard multiple resistance present was detected to FBZ, PYR and IVM. Macrocylic lactones proved to be the most effective drugs, with some evidence of resistance to IVM and highest activity of MOX, despite a single case of reduced efficacy in Germany. These data call for the development and implementation, among practitioners, owners and managers, of further plans to reduce the expansion of the anthelmintic resistant populations and to use those anthelmintics that remain effective in a manner that preserves their efficacy as long as possible.