Long-term affected flat oyster (Ostrea edulis) haemocytes show differential gene expression profiles from naïve oysters in response to Bonamia ostreae.

European flat oyster (Ostrea edulis) production has suffered a severe decline due to bonamiosis. The responsible parasite enters in oyster haemocytes, causing an acute inflammatory response frequently leading to death. We used an immune-enriched oligo-microarray to understand the haemocyte response to Bonamia ostreae by comparing expression profiles between naïve (NS) and long-term affected (AS) populations along a time series (1 d, 30 d, 90 d). AS showed a much higher response just after challenge, which might be indicative of selection for resistance. No regulated genes were detected at 30 d in both populations while a notable reactivation was observed at 90 d, suggesting parasite latency during infection. Genes related to extracellular matrix and protease inhibitors, up-regulated in AS, and those related to histones, down-regulated in NS, might play an important role along the infection. Twenty-four candidate genes related to resistance should be further validated for selection programs aimed to control bonamiosis.

Skeletal anomalies in reared Senegalese sole Solea senegalensis juveniles: a radiographic approach.

Reared Senegalese sole Solea senegalensis Kaup show a high incidence of vertebral anomalies; however, little is known about its skeletal anomaly profile in the later farming phases. The purpose of this study was to provide a detailed description and quantification of the most common skeletal anomalies in reared Senegalese sole in the juvenile stage by means of computed radiography. A total of 374 Senegalese sole were classified according to the external morphology of the fish as normal or altered and then radiographed in latero-lateral and in dorso-ventral projections. Radiographic evaluation of anomalies focused especially on vertebral body anomalies (VBA) and vertebral column deviations (VCD). The 2 orthogonal projections provided a more complete visualization of the skeleton. Approximately 75% of the individuals showed at least 1 anomaly, while VBA and/or VCD were detected in 48.9% of the specimens. Regarding external morphology, 88% of the fish were categorized as normal, although about 72% of these normal fish displayed abnormalities in radiographies. The most frequent anomalies consisted of deformations of the caudal complex plates (hypurals, parhypural and epural), preurals and caudal vertebrae. Scoliosis was the most prevalent among VCD, affecting the caudal area in almost 15% of the individuals. The anomaly profile at the juvenile stages showed some differences compared to what has been reported previously in earlier stages of development. In light of these results, further investigation into the progression of skeletal anomalies over time and the causative factors at later stages is required.

New insight on vertebral anomalies in cultured Senegalese sole (Solea senegalensis, Kaup) at early stages of development.

Senegalese sole (Solea senegalensis, Kaup) is a promising flatfish species in aquaculture. However, skeletal anomalies are still a great concern in sole farming. Investigation of this issue is crucial to improving larval quality and optimizing production. The aim of this study was to thoroughly assess anomalies in the rachis of reared sole at early developmental stages. Sole (n = 507) were sampled at 31 or 32 days after hatching (dah). The specimens were stained with alcian blue and alizarin red and evaluated for the detection of vertebral deformities. Most fish presented 9:34:3 vertebrae in abdominal, caudal and caudal complex regions, respectively. Remarkably, all specimens showed at least one spinal anomaly. Alterations of neural/haemal elements, as well as deformities of hypurals, parhypural and epural, were recurrent. Vertebral body anomalies and/or vertebral column deviations were identified in 52% of the individuals. Vertebral deformations and fusions were common, especially in caudal complex. 'Minor' anomalies were predominant, and some of the detected disorders might be a result of non-/low-pathological processes. These results contribute a new insight into the main skeletal anomalies affecting cultured sole larvae. Further research is required to determine their impact on fish welfare and external appearances at commercial stages.

Effects of Enteromyxum scophthalmi experimental infection on the neuroendocrine system of turbot, Scophthalmus maximus (L.).

Enteromyxum scophthalmi is an intestinal myxosporean parasite responsible for serious outbreaks in turbot Scophthalmus maximus (L.) culture, in North-western Spain. The disease affects the digestive tract, provokes severe catarrhal enteritis, emaciation and high rates of mortality. The digestive parasitization triggers a response with the coordinate participation of immune and neuroendocrine systems through the action of peptides released by enteroendocrine cells and present in nervous elements, acting as neuro-immune modulators. The present study was designed to assess the response of the turbot neuroendocrine system against E. scophthalmi infection. Immunohistochemical tests were applied to sections of the gastrointestinal tract of uninfected and E. scophthalmi-infected turbot to characterize the presence of bombesin (BOM), glucagon (GLUC), somatostatin (SOM), leu-enkephalin (LEU) and met-enkephalin (MET). The occurrence of E. scophthalmi in the turbot gastrointestinal tract increased the number of enteroendocrine cells immunoreactive to SOM, LEU and MET. On the other hand, BOM and GLUC immunoreactive cells were less numerous in the gastrointestinal tract of the parasitized turbot. Scarce immunoreactivity to BOM, GLUC and SOM was observed in nerve fibres and neurons of the myenteric plexus of control and infected fish. The results indicate that E. scophthalmi infection in turbot induced changes in the neuroendocrine system, with the diminution of the anorexigenic peptides BOM and GLUC; the increase of enkephalins, related to pro-inflammatory processes; and the increase of SOM, which may cause inhibitory effects on the immune response, constituting a compensatory mechanism to the exacerbated response observed in E. scophthalmi-infected turbot.

Immunohistochemical diagnosis of tenacibaculosis in paraffin-embedded tissues of Senegalese sole Solea senegalensis Kaup, 1858.

A sensitive and specific immunohistochemical technique was developed to improve the diagnosis of tenacibaculosis and to better understand its pathogenesis. Senegalese sole Solea senegalensis Kaup, 1858 were inoculated subcutaneously with a bacterial suspension of Tenacibaculum maritimum, and samples were taken at different hours post-inoculation. Sections from different organs were used as positive controls. In addition, a total of 128 field samples from different organs collected from tenacibaculosis outbreaks were used. Tenacibaculum maritimum antigens were detected in several organs of experimentally infected Senegalese sole and in at least one of the tissues from fish suffering from natural tenacibaculosis previously confirmed by culture and PCR-based methods. In fish collected during outbreaks, a strong positive reaction was detected in ulcerative skin areas. Moreover, bacterial antigen was identified inside scale pockets and in sites of the skin with mild lesion. In kidney and spleen, evident immunostaining of bacterial antigen was detected in both naturally and experimentally infected fish. Besides, the presence of T. maritimum in the intestinal tract without associated histological changes suggests that this organ may act as a reservoir for T. maritimum. The results of this study confirm the usefulness of IHC for the diagnosis of tenacibaculosis in paraffin-embedded tissues.

Study of the distribution of active caspase-3-positive cells in turbot, Scophthalmus maximus (L.), enteromyxosis.

Enteromyxosis caused by Enteromyxum scophthalmi is one of the parasitizations with a higher economic impact on turbot, Scophthalmus maximus (L.), aquaculture. This myxosporean produces severe catarrhal enteritis with abundant inflammatory infiltrates in the lamina propria-submucosa (LP), epithelial detachment and leucocyte depletion of the lymphohaematopoietic organs. Some advances made on the pathogenesis pointed to a role of apoptosis in the enteromyxosis. Therefore, the main aim of this work was to employ the TUNEL assay and the anti-(active caspase-3) immunohistochemical assay to detect apoptotic cells in both healthy and E. scophthalmi-infected turbot in order to establish the presence and distribution of apoptotic cells during development of the disease. More apoptotic cells located within the gastrointestinal epithelium were observed in the initial stages of the infection in E. scophthalmi-infected turbot compared with non-infected turbot. As the infection progressed, a higher degree of apoptosis occurred in the epithelium of folds heavily parasitized. In the severely infected turbot, apoptosis was also found among the leucocytes of the intestinal inflammatory infiltrates. Moreover, the number of active caspase-3-positive cells in the lymphohaematopoietic organs tended to increase with disease severity. In view of the results, increased apoptosis in the epithelium may favour the scaling that occurs during enteromyxosis and cell death of leucocytes in the intestinal LP, contributing to leucocyte depletion in severe cases.

Quantitative and qualitative evaluation of iNOS expression in turbot (Psetta maxima) infected with Enteromyxum scophthalmi.

Enteromyxum scophthalmi is the causative agent of turbot enteromyxosis, an intestinal parasitisation that produces severe desquamative enteritis leading to a cachectic syndrome and eventually the death. It is well known the importance of the innate immune response against parasites in fish, with the release of antimicrobial substances such as reactive oxygen and nitrogen species, produced by the inducible nitric oxide synthase (iNOS). This enzyme is mainly found in phagocytes, but also in structural cells from the intestinal mucosa. The aim of this study was to characterize iNOS in intestine and lymphohaematopoietic organs (spleen and anterior kidney) of turbot by means of immunohistochemistry in order to assess the possible changes of this enzyme through the infection. The presence of the enzyme was evaluated in control and E. scophthalmi-infected turbot. The results showed immunoreactivity in the apical border of enterocytes and mild staining of goblet cells in both control and infected turbot although it was more evident and widespread in infected turbot compared to control. Moderate numbers of iNOS+ cells were present in the lamina propria-submucosa of fish which presented moderate and severe inflammatory infiltrates at this level. In spleen and kidney, iNOS+ cells were scattered through the parenchyma and, in severely infected fish, tended to be allocated near the vascular structures and melano-macrophage centres. The number of positive cells at the lymphohaematopoietic organs was significantly higher in infected turbot and increased as infection progressed. The increase in the expression of iNOS in the tissues of E. scophthalmi-infected turbot was more evident in individuals with severer lesions. The measurement of the levels of iNOS during turbot enteromyxosis reveals a possibly delayed response that would not able to eliminate the parasites but would exacerbate mucosal injury.

Morphopathological features of a severe ulcerative disease outbreak associated with Tenacibaculum maritimum in cultivated sole, Solea senegalensis (L.).

This study describes morphopathologic changes in naturally infected farmed Senegalese sole affected by tenacibaculosis caused by Tenacibaculum maritimum. Macroscopic observation, in addition to light microscopy and scanning electron microscopy, was used to study the lesions. Main lesions were characterized by complete loss of epidermis and dermis, as well as extensive necrosis of muscle layers. Mild-to-moderate inflammatory response with the presence of macrophages was noted around hyaline degenerated muscle cells. Gram-negative filamentous bacteria could be detected only at the dermis. Under scanning electron microscopy, filamentous bacteria located over the scales without epithelium could be observed. These findings together with the isolation and PCR detection of the bacteria in kidney and skin tissues suggest that once the bacteria reach the dermis, probably through eroded epidermis, they are able to proliferate and produce enzymes that are responsible for the damage in the underlying tissues.

First description of an olfactory neuroblastoma in goldfish Carassius auratus: a case report.

An external pinkish growing mass that emerged from the right nostril of an adult goldfish Carassius auratus L. was evaluated by means of light microscopy and immunohistochemistry. The neoplasm presented a well-developed fibrovascular stroma associated with solid cell nests and a large number of Flexner-Wintersteiner rosettes. Myelinated fibres were observed around them. Neoplastic cells showed a prominent degree of nuclear atypia and low mitotic activity. The latter was in agreement with the low reactivity of tumour cells to anti-proliferating cell nuclear antigen antibody. Immunohistochemistry also revealed anti-neuronal nitric oxide synthase, anti-S100 protein, antineuropeptide Y, and anti-cytokeratin immunoreactivity in tumour cells as well as in normal olfactory epithelium of goldfish control sections. Histopathological and immunohistochemical findings strongly suggest a diagnosis of an olfactory neuroblastoma (ONB). To our knowledge this is the first description of ONB in goldfish.

Mucosal CD3ε+ cell proliferation and gut epithelial apoptosis: implications in rainbow trout gastroenteritis (RTGE).

Rainbow trout gastroenteritis (RTGE) is an emerging disease that has acquired new relevance in European rainbow trout, Oncorhynchus mykiss (Walbaum), culture, because of the economic losses it causes. Disease aetiology and pathogenesis remain unclear. The lesions appear restricted to the gastrointestinal tract where extensive mucosal detachment associated with high numbers of segmented filamentous bacteria (SFB) can be detected. In this study, an RTGE outbreak in north-western Spain was investigated, and findings observed in diseased trout were compared with control fish. PAS stain and immunohistochemical assays with anti-CD3ε and anti-active caspase-3 antibodies were performed. The results showed that CD3ε+ inflammatory infiltrates were present in the intestine of diseased trout both in the lamina propria-submucosa and within the epithelium. Moreover, an increased number of caspase-3+ cells in the intestinal mucosa and also strong anti-caspase-3 immunoreactivity in desquamated cells in the gut lumen were observed. Changes in the number of goblet cells were also found, resulting in an increase or depletion of mucous cells depending on the severity of the intestinal lesions. These findings suggest that T cells and apoptosis play an important role in the development and pathogenesis of RTGE.

An immunohistochemical study of the gut neuroendocrine system in juvenile pejerrey Odontesthes bonariensis (Valenciennes).

In this study, several neuropeptides were identified by immunohistochemistry in neuroendocrine cells (NEC) located in the gut epithelium and nerve cell bodies of the enteric nervous system of pejerrey Odontesthes bonariensis, a species that is a promising candidate for intensive aquaculture. The neuropeptides involved in orexigenic or anorexigenic action, i.e. gastrin, cholecystokinin-8, neuropeptide Y and calcitonin gene-related peptide (CGRP), displayed a significantly higher number of immunoreactive NECs in the anterior intestine, suggesting that this region of the gut plays an important role in the peripheral control of food intake. On the other hand, leu-enkephalin and vasoactive intestinal peptide (VIP), both associated with the modulation of the enteric immune system, showed no significant variations in the mean value of immunopositive NECs between the anterior and posterior intestine. This may indicate that their activity is required at a similar level along the entire gut. In addition, CGRP and VIP-immunoreactive neurons and nerve fibres were observed in the myenteric plexus, which might exert synergistic effects with the neuropeptides immunolocalized in NECs.

Light and electron microscopic studies on turbot Psetta maxima infected with Enteromyxum scophthalmi: histopathology of turbot enteromyxosis.

In the last decade, a new parasite that causes severe losses has been detected in farmed turbot Psetta maxima (L.), in north-western Spain. The parasite was classified as a myxosporean and named Enteromyxum scophthalmi. The aim of this study was to characterize the main histological changes that occur in E. scophthalmi-infected turbot. The parasite provoked catarrhal enteritis, and the intensity of the lesions was correlated with the progression of the infection and with the development of the parasite. Infected fish were classified into 3 groups, according to the lesional degree they showed (slight, moderate and severe infections). In fish with slight infections, early parasitic stages were observed populating the epithelial lining of the digestive tract, without eliciting an evident host response. As the disease progressed, catarrhal enteritis was observed, the digestive epithelium showed a typical scalloped shape and the number of both goblet and rodlet cells was increased. Fish with severe infections suffered desquamation of the epithelium, with the subsequent release of parasitic forms to the lumen. The dislodged enterocytes underwent anoikis, a mode of apoptosis triggered by the loss of anchorage, which might facilitate spreading of the parasite. Lymphohaematopoietic depletion was also observed, mainly in head kidney and spleen, which could contribute to the high virulence of this parasite.

Development of rodlet cells in the gut of turbot (Psetta maxima L.): relationship between their morphology and S100 protein immunoreactivity.

Rodlet cells are an enigmatic cell type described in tissues of both marine and freshwater teleosts. Although their structure is well established, up to date their function remains subject of debate. However, there is consensus among the majority of researchers that rodlet cells play an important role within immune system, and this function is probably related with the release of rodlets due to contractile capability of their fibrous layer. Regulation of the contraction mechanism would require proteins that modulate Ca(++) intracellular concentration to be expressed in rodlet cells. We performed a morphological and immunohistochemical study at light and electron microscopy levels to assess S100 protein immunoreactivity in developing rodlet cells. Immature stages did not exhibit immunoreactive signal; however, immunoreactivity was observed in the fibrous layer of both transitional and mature rodlet cells. The latter stage also showed immunosignal within the rodlets. These findings suggest a clear association between S100 protein expression and rodlet cell development that could be linked to the regulation of rodlet activity and contractile property of their fibrous layer. Furthermore, S100 protein antibody constitutes a novel marker for rodlet cells that could be used in future studies of this particular cell type.

Toxoplasmosis in Bennett's wallabies (Macropus rufogriseus) in Spain.

Toxoplasmosis is one of the more common parasitic zoonoses world-wide. In this study, an epizootic of toxoplasmosis among captive Bennett's wallabies (Macropus rufogriseus) from different locations is reported. By means of light microscopy, Toxoplasma gondii-like tachyzoites were observed associated to interstitial pneumonia, non-suppurative myocarditis, cholangiohepatitis and severe gastroenteritis. The protozoa stained positively with a T. gondii antibody and ultrastructurally were similar to T. gondii. Strikingly, tachyzoites appeared sometimes in an intranuclear location within granulocyte-like cells. Feral cats or reactivation of a latent infection are discussed as the possible sources of infection. As far as we know, this is the first confirmed report of toxoplasmosis in Bennett's wallabies in Spain and Europe, and may constitute a risk of infection for humans since new alimentary habits are being imposed in our countries.

Levamisole activates several innate immune factors in Scophthalmus maximus (L.) Teleostei.

Levamisole, originally synthesized as an anti-helminthic, has been widely used in human and veterinary medicine as an immunomodulator or adjuvant. However, data on its use in fish are scarce, and no information is available for turbot. To study the effects of levamisole treatment on the innate immune system of juvenile turbot, two different doses (D1=500 mg/kg; D2=250 mg/kg dry food) were orally administered for 2 weeks and samplings were performed at -10, 14, 28, 49 and 77 days post treatment (p.t.). Biometrical, haematological, histological and immunological data were obtained. Specific growth rate was higher in the medicated groups than in the control (C), but the difference was statistically significant only for D1 fish at day 49 p.t. The leucocytes/thrombocytes ratio was significantly higher in D1 than in C fish at 14 days p.t., but decreased subsequently. At most samplings, the percentage of the circulating lymphocytes was lower and that of the progranulocytes was higher in the medicated fish than in the C ones. The percentage of fish with high haemotopoietic activity in the kidney was clearly higher in D1 and D2 fish than in C ones at some sampling points. The respiratory burst activity of blood leucocytes was significantly higher in D1 fish than in C ones in all samplings, except at day 77 p.t. when control fish experienced a rebound effect. In all medicated fish, an initial increase of such activity was observed, followed by a further decrease. Their serum peroxidases followed a contrary pattern, with a decrease in the second sampling and a subsequent and non-significant recovery, a situation also observed for serum lysozyme and complement. Therefore, oral levamisole treatment actually affects some turbot immune factors, although stimulation or depression can occur depending on the considered factor and the administered dose. These results point out the interest of further studies on the mechanisms involved in the levamisole action for its adequate use as immunomodulator.

Temporary inhibition of neuronal apoptosis in Aujeszky's disease virus-infected swine.

Previous studies have shown that during acute infection of the porcine trigeminal ganglia (TG), Aujeszky's disease virus (ADV)-infected neurons are protected from apoptosis induced by the virus itself and by cells of the immune system. However, TG neurons productively infected by ADV finally die and are phagocytosed by adjacent cells, a fact that leads us to speculate that the inhibition of neuronal apoptosis by ADV may be temporary rather than absolute. To address this issue we used TG and brain stem from pigs during acute infection by ADV. Infected cells were detected by immunohistochemical staining of viral antigens, whereas apoptotic cells were identified with an anti-active caspase-3 antibody, the TUNEL assay and by transmission electron microscopy. The results obtained in this study support the contention that the inhibition of neuronal apoptosis by ADV is temporary, since activation of caspase-3 could be detected in infected neurons at late stages in infection and because foci of advanced neuronophagia contained neurons exhibiting typical ultrastructural features of apoptosis.

Meningoencephalitis associated with Staphylococcus warneri in a dog.

A case of meningoencephalitis in a dog caused by Staphylococcus warneri is reported here. The history and clinical signs were suggestive of possible central nervous system infection. Analysis of cerebrospinal fluid documented a neutrophilic pleocytosis (890 cells/mul) and the presence of occasional intracellular cocci. Staphylococcus warneri was isolated from the microbiological culture of the cerebrospinal fluid. Treatment consisted of intravenous antibiotics, supportive care and anticonvulsants for the generalised seizures that developed after admission. Histological assessment confirmed the location and extension of bacterial meningoencephalitis. Thrombotic meningoencephalitis associated with Staphylococcus warneri infection has not, to the authors' knowledge, been previously reported in dogs.

Diagnosis of atoxoplasmosis in a canary (Serinus canarius) by histopathologic and ultrastructural examination.

Our paper describes pathologic and ultrastructural features of a canary (Serinus canarius) infected by Atoxoplasma sp. Histologically, numerous mononuclear cells were observed in intestinal mucosa, liver, and spleen. Most of these cells contained a variable number of protozoal-like organisms. These organisms were identified as Atoxoplasma sp. by ultrastructural examination of reprocessed paraffin-embedded tissues.

Squamous cell carcinoma of the oropharynx and esophagus in a Japanese bantam rooster.

Squamous cell carcinoma of the oropharynx and esophagus was diagnosed in an adult Japanese bantam rooster. Grossly, a cauliflowerlike mass with irregular edges was found involving the ventrolateral surfaces of the caudal portion of the oropharynx and cervical portion of the esophagus. The large volume of the mass almost occluded the lumen of the alimentary passage. Histologically, the tumor consisted of irregular cords of pleomorphic epithelial cells that showed a disorganized pattern of growth and invaded the adjacent tissues. Keratinized epithelial cells and moderate numbers of keratin pearls were readily observed. The mitotic index was low, and, although the tumor was locally invasive, we found no evidence of vascular invasion or metastasis.

Cryptosporidium scophthalmi n. sp. (Apicomplexa: Cryptosporidiidae) from cultured turbot Scophthalmus maximus. Light and electron microscope description and histopathological study.

Cryptosporidium scophthalmi n. sp. is described from the turbot Scophthalmus maximus L., sampled from different farms on the coast of NW Spain. The parasite was found mainly in the intestinal epithelium and very seldom in the stomach. Oocysts were almost spherical, with 4 naked sporozoites and a residuum, and measured 3.7-5.03 x 3.03-4.69 microm (mean 4.44 x 3.91) (shape index 1.05-1.34, mean 1.14). Sporulation was endogenous, as fully sporulated oocysts were found within the intestinal epithelium, lumen and faeces. Merogonial and gamogonial stages were in the typical extracytoplasmic position, whereas sporogonial stages were deep within the epithelium. Oocysts and other stages of C. scophthalmi comply with most of the diagnostic features of the genus Cryptosporidium, but differ from all hitherto described species. Ultrastructural features, including the characteristic feeding organelle, were mainly comparable with those of other Cryptosporidium species. Mitochondria were frequently observed in sporozoites. Infection prevalence was very variable, and juvenile fish were most frequently and intensively parasitised. External clinical signs were not detected, although some fish showed intestinal distension at necropsy. The marked histopathological damage occurring in severe infection includes distension of epithelial cells by large vacuoles, containing clusters of oocysts, and can lead to sloughing of epithelial cell remnants and oocysts or even detachment of intestinal mucosa. An inflammatory reaction involving leucocyte infiltration was sometimes observed.