The versatility of simplicity: Structures of Cardiocephaloides longicollis used for different purposes during cercarial transmission.

Transmission and infection strategies are critical for completing the life cycles of trematode parasites, which are characterized by complex life cycles involving multiple hosts and stages. Transmission between the first and second intermediate hosts typically relies on cercariae, a free-swimming larval stage that displays a series of behaviors to efficiently disperse, locate, attach to, and infect the next host. The aim of this study is to provide detailed information on behaviors used by furcocercariae (bifurcated tail) during its transmission from the snail to the fish host, using the laboratory-established model of Cardiocephaloides longicollis (Strigeidae). These cercariae are released from snails into seawater, where they swim, locate, penetrate the skin of fish, and encyst as metacercariae in their brain. In a series of in vivo assays, freshly-emerged cercariae were used to visually study their behavior and locomotion. Histopathology of experimentally infected gilthead seabreams with C. longicollis, taken at sequential post-infections times, were analysed to localize the migrating cercariae to the fish brain. Our results show that simplicity and versatility are the key features for the success of cercariae transmission by using their organs for different purposes. While 80% of the behavior was spent in a resting position, the most common swimming behavior was with tail-first, which is commonly described in furcocercariae to reach the host microhabitat. However, C. longicollis relies more on the furcae of the tail by using them as a propeller providing thrust and guidance when they swim, instead of using the tail stem. After attaching to the fish skin, cercariae rapidly creep on it using the oral- and ventral-suckers simulating a leech-like movement until they find a suitable penetration site. To penetrate, cercariae press the cephalic structures against the skin, while the ventral sucker anchors the cercariae to it. After this, they switch their locomotion to a slow peristaltic movement, opening the path through tissues with the help of their cephalic structures and anchoring their body with their surface spines. This is consistent with the post-penetration histological analyses, which suggested that C. longicollis cercariae move between the cells of the connective tissue and muscle fibers when migrating towards the fish's brain, without provoking relevant tissue damage or host responses. Understanding the versatility of cercarial structures to adapt to external conditions enriches our knowledge on parasites and their transmission ecology, opening the door to the design of avoidance methods in fish farms struggling with harmful parasites.

Horizontal transmission of Endolimax piscium, causative agent of systemic amoebiasis in Senegalese sole Solea senegalensis.

Systemic amoebiasis of Senegalese sole Solea senegalensis is caused by Endolimax piscium Constenla, Padrós & Palenzuela, 2014 a cryptic parasitic member of the Archamoebae whose epidemiology is yet unknown. To test whether the parasite can be transmitted horizontally, an experimental trial by cohabitation between non-infected and infected fish was designed. Transmission of the parasite from naturally infected to healthy fish was confirmed in the experiment, with the water as the most likely route of infection. Under the conditions of the study, the infection process was remarkably slow, as parasites could be detected by in situ hybridization within the intestinal mucosa of recipient fish only after 17 wk of cohabitation, and none of the new hosts displayed clinical signs of disease. Long prepatent period and the need for additional triggering factors for the development of the clinical condition are suggested. The intestinal mucosa is proposed as the tissue where the amoeba can survive as endocommensal, but also as an invasion route from which the parasite would disperse to other organs.

Histopathological characterisation of retinal lesions associated to Diplostomum species (Platyhelminthes: Trematoda) infection in polymorphic Arctic charr Salvelinus alpinus.

The eye represents an immune privileged organ where parasites can escape host reactions. This study provides the first systematic evidence of the pathology associated with Diplostomum sp. infection in the eye retina of fish (i.e. Arctic charr). Histological sections showed that the trematodes caused mechanical disengagement between the retinal pigmentary epithelium and the neurosensory retina, with damaged cones and rods in the outer segment and epithelium reduced to a single layer of pigmentary cells. The metacercariae were "floating" in possibly fluid-filled vesicles together with several round cells, mostly located in the anterio-dorsal and anterio-ventral areas of the eye near the iris. The round cells may indicate internal retinal damage repair mechanisms, without connections to the general immune system. Metacercariae intestines contained pigmented cellular debris indicating that they feed on retinal epithelium. These retinal lesions may have similar vision effects as focal retinal detachment in vertebrates. Diplostomum metacercaria alters fish visual acuity but may in a lesser degree lead to a severe or total visual impairment because of repairing mechanisms. The pathology in the retina seems thereby to be dependent on fish size, age and dose.

Hamatum osteoblastom / Osteoblastoma del hueso ganchoso.

Osteoblastoma is an infrequent primary osseous tumour. Its presentation in the upper extremities and more specifically in the carpal bones is unusual. We present a case of osteoblastoma localized in the hamate bone and review the treatment realized in this infrequent localization. A young male patient with pain and swelling in the back of his hand of one year's evolution, resistant to medical treatment. Complementary tests showed lytic tumefaction in the hamate bone with non-aggressive characteristics. It was treated by curettage and filling the iliac crest with autologous graft. The pathological anatomical study diagnosed that it was a case of osteoblastoma. The result was satisfactory, with total disappearance of the pain and a radiological image of complete restitution of the osseous defect, with no signs of recurrence after 4 years. The treatment should be curettage plus autologous graft. Conversely, resection of the affected bone can be considered in cases with aggressive data.

Development of different diagnostic techniques for Endolimax piscium (archamoebae) and their applicability in Solea senegalensis clinical samples.

Systemic amoebiasis of sole is caused by Endolimax piscium, a cryptic parasitic archamoeba whose epidemiology and pathogeny are yet unknown. To establish reliable detection methods for this parasite, a battery of molecular diagnostic tools (ISH, PCR and qPCR) were developed and evaluated with a panel of clinical samples from symptomatic diseased fish and from apparently normal animals of different stocks. As there is neither enough background information on the epidemiology of the disease nor a validated reference method, comparison of tests used a composite reference method approach. The ISH technique was the most specific and sensitive in intestine samples and particularly useful as a reference confirmatory method, while the best method in muscle samples was qPCR. Application of the tests to asymptomatic fish demonstrated presence of parasites in a large proportion (>25%) of their intestines, suggesting that this is the point of entry of the amoebae and the initial stage in the development of the disease. The triggering factors that facilitate the breaching of the intestinal barrier by E. piscium, causing granulomatous lesions in other organs and systemic spreading, are not completely understood but our results point to the connective tissue as a preferential target for parasite development and migration.

[Treatment of recalcitrant humeral nonunion in elderly patients]. / Tratamiento de la pseudoartrosis recalcitrante de húmero en pacientes de edad avanzada.

BACKGROUND: Humeral nonunion that persists through various unsuccessful attempts at management, presents a real challenge. When combined with severe osteoporosis in elderly patients, the problems only increase. This study aims to investigate the best option for treating this combination of factors. MATERIALS AND METHOD: This study analyses the outcomes of five patients with the following inclusion criteria: nonunion of the humeral diaphysis, after at least two unsuccessful interventions and/or after a period of 18 months or more, associated with severe osteoporosis in patients aged over 65. All cases were treated by debridement of the site of nonunion and stabilisation with locking plates and bone graft. RESULTS: All cases consolidated after a mean follow-up period of 19 months (8-36 months). CONCLUSIONS: In cases of humeral nonunion in elderly patients with osteoporotic bones, locking plates have been proven to provide consistent, long-term stability, with better results than traditional compression plates. The authors consider that locking plates should be considered as a valid option in cases of extreme bone and biological instability, as is the case in recalcitrant humeral nonunion.

Endolimax piscium sp. nov. (Amoebozoa), causative agent of systemic granulomatous disease of cultured sole, Solea senegalensis Kaup.

A new amoeba species pathogenic for Senegalese sole is described based on ultrastructural analysis and SSU rDNA phylogenetic inference. The parasite presents round to ovoid trophozoites (<5 µm) with a high degree of intracellular simplification. No mitochondria were observed, but mitosome-like organelles were present. No cysts could be detected. Phylogenetic analysis confirmed the Senegalese sole parasite as an amitochondriate Archamoeba related to Endolimax nana and Iodamoeba spp., and we tentatively describe it as a new species in the genus Endolimax, Endolimax piscium. However, the genetic distance with E. nana is quite large, with only 60% pairwise identity between both SSU rDNA genotypes. Although the overall topology of the Archamoebae cladograms containing E. piscium was consistent, the support for the branching of Endolimax spp. relative to its closest neighbours was variable, being higher with distance or parsimony-based inference methods than with ML or Bayesian trees. The use of stringent alignment sampling masks also caused instability and reduced support for some branches, including the monophyly of Endolimax spp. in the most conservative data sets. The characterization of other Archamoebae parasitizing fish could help to clarify the status of E. piscium and to interpret the large genetic distance observed between Endolimax species.

Parasitisation by Bathycreadium elongatum (Digenea, Opecoelidae) in pyloric caeca of Trachyrincus scabrus (Teleostei, Macrouridae).

A novel process of transmural passive displacement of a digenean parasite was studied in the digestive tract of the roughsnout grenadier Trachyrincus scabrus, which is found in the northwestern Mediterranean Sea. This mechanism seems to facilitate the elimination of a significant portion of intestinal parasites. The digenean parasite Bathycreadium elongatum was found in the intestine, mainly within pyloric caeca, in 74.4% of T. scabrus, with a mean abundance of 44 individuals per fish. Nodule-like lesions were also found in the mesentery of pyloric caeca of infected T. scabrus. Histological sections of the nodules revealed granulomatous inflammatory responses surrounding degraded digeneans. Partial nucleotide sequences of the 28S rRNA gene obtained from intracaecal B. elongatum and from the core of the nodules of the mesentery of pyloric caeca showed 100% mutual identity with an overlap of 971 bp. The greatest abundance of both intracaecal B. elongatum and nodules occurred in spring. During summer, and especially autumn, the abundance of intracaecal B. elongatum decreased. Prevalence and abundance of nodules increased in autumn. In winter intracaecal parasite abundance and prevalence began to increase, but decreased again in nodules. During spring and summer, parasites pass into the visceral cavity, hypothetically owing to the fragility of the wall of pyloric caeca in their apical zone, and become degraded through a granulomatous inflammatory response. This process seems to have a detrimental effect on the B. elongatum cycle since some of parasites are trapped and degrade in the connective tissue in which they are unable to complete their life cycle.

Morphological and histological study of larval development of the Senegal sole Solea senegalensis: an integrative study.

This study provides a comprehensive description of the main morphological and histological events that take place during larval and post-larval development of Senegal sole Solea senegalensis in order to establish a reference for its normal developmental organogenesis. Five stages have been described. Before gill development at the onset of metamorphosis (eye migration process, stage 4c), the skin was the main site of gas and ion exchange, whereas during stages 3 and 4, the skin begins differentiating into the definitive juvenile structure. The timing of development of the endocrine system depends on each organ, the endocrine pancreas and thyroid gland being the first to differentiate (stages 2 and 3, respectively), followed by the interrenal tissue and stannius corpuscles that develop at metamorphosis (stages 4 and 4c, respectively). The differentiation and maturation of the lymphohaematopoietic organs was coupled with the increase in complexity of the cardiovascular system and the presence of mature erythrocytes (stage 4b), which might be attributed to the change in respiration and the development of fully functional gills. In the differentiation of sensory structures, the development of eyes, inner ear, neuromasts and olfactory organs was rapid, with most of these organs becoming fully developed soon after hatching (stage 1). Vision, chemo- and mechano-reception developed very early in ontogeny, in parallel with the development of the central nervous system and changes in feeding habits. Although the general pattern of development in S. senegalensis appeared similar to most marine fish larvae already described, there were species-specific ontogenetic characteristics probably derived from the species' particular environment (subtropical waters) and behaviour (nocturnal, benthic, omnivorous feeding habits). These results on the organogenesis of larvae are a useful tool for establishing the functional systemic capabilities and physiological requirements of larvae to ensure optimal welfare and growth under aquaculture conditions.

Histopathological and ultrastructural studies on a novel pathological condition in Solea senegalensis.

A new parasitic disease affecting cultured sole Solea senegalensis (Kaup, 1858) is characterised by the presence of external protuberances in the skin of the affected fish. These lesions correspond to nodules in the muscular tissue showing an abscess-like aspect. Similar lesions were found in the kidney, heart, liver and digestive tract. Histological sections of these nodules revealed the presence of a large core formed mainly of necrotic tissue surrounded with fibroblasts and macrophages. Round-shaped plasmodial organisms were found in the external layer of the nodules and usually inside macrophages or fibroblasts. These organisms were also observed in the intestinal mucosa inside phagocytic cells or parasitophorous vacuoles within the enterocytes. The morphological and ultrastructural characteristics of these organisms are similar to the morphology of some groups of parasites described as fish pathogens. The main features suggest that these organisms could be amoebae or parasites with an amoeboid or plasmodial form in their developmental cycle.

Microbial and histopathological study of the vibriosis caused by Vibrio vulnificus serovar E in eels: the metalloprotease Vvp is not an essential lesional factor.

Vibrio vulnificus biotype 2 serovar E (Bt2-serE) is a zoonotic pathogen that causes a haemorrhagic septicaemia in eels, called warm water vibriosis. The main objective of the present work was to study the onset of the eel vibriosis from the microbiological and histopathological viewpoint, as well as to ascertain the role of the protease Vvp as a lesional factor by comparing the histopathological lesions caused by the wild strain and its vvp deficient derivative. The wild-type strain was observed to attach to the gills, where it multiplied following saturation dynamics, subsequently invading the blood stream and reaching the internal organs. Here it reached population sizes that are notably lower than those associated with other fish septicaemia. Parallel to bacterial growth, there was a notable decrease in haematocrit values and haemoglobin concentration in blood as well as extensive haemorrhages in all the analysed organs. The main histopathological lesions were detected in the head kidney in the form of extensive necrosis affecting the haematopoietic tissue. Very few bacteria were visualized in the different organs, most of which were close to blood cells and capillary vessels, which is compatible with the results obtained in the microbiological study. The same lesions were produced when extracellular products (ECPs) were injected instead of bacteria or when the vvp-defective mutant or its ECPs were injected. The overall results suggest that the pathology caused by V. vulnificus in the eel is not caused by massive bacterial growth in the blood and internal organs but, rather, by the effect of potent toxic factors other than the metalloprotease, which have yet to be determined.

Histopathology of experimental scuticociliatosis in turbot Scophthalmus maximus.

A scuticociliate strain (B-2), originally isolated from an outbreak in a turbot Scophthalmus maximus (= Psetta maxima) farm in Galicia (northwestern Spain) and maintained in axenic culture, was injected intracoelomically (lethal dose 80 equivalent, LD80) in healthy turbot (50 g). Ciliate-injected fish were kept under controlled conditions in a recirculating seawater system and sampled on Days 1 through 8, 10, 12 and 14 postinfection (PI). Necropsies were conducted and included blood collection from the caudal vein and samples of liver, spleen, heart, digestive tract, kidney, gills, abdominal wall and neurocranium taken for routine histology. Mortality occurred from Day 6 until Day 12 PI and reached 66.7% by the end of the experiment. Presence of ciliates in the coelomic fluid was scarce until Day 4 PI. Parasitaemia was only observed from Day 5 until Day 10 PI and its incidence was always low. Presence of scuticociliates in tissue sections followed a progressive pattern of diffusion, with ciliates showing preference for loose connective tissue and also a clear haematophagous activity. The most severely affected organs were the pancreas and digestive tract. No special tropism for nervous tissues was observed in this study. The inflammatory reaction was variable depending on the tissue. After 3 wk, survivors had apparently managed to extinguish the infection.

Sharpsnout sea bream (Diplodus puntazzo) humoral immune response against the parasite Enteromyxum leei (Myxozoa).

The humoral innate immune response of sharpsnout seabream Diplodus puntazzo against the myxozoan Enteromyxum leei was studied. Enteromyxosis was transmitted by cohabitation and a group of uninfected fish served as control. At 5, 12, 19, 26, 40 and 55 days post-exposure (p.e.), control and recipient fish were sampled to determine the prevalence of infection and some humoral innate immune parameters (antiprotease, antitumoral and peroxidase activities). Prevalence of infection was high from day 12 p.e. and reached 100% at days 40 and 55, when intensity of infection was medium to severe. The antiprotease activity was significantly increased in E. leei-exposed fish with respect to control fish at days 12 and 19 p.e. The serum antitumoral activity was slightly lower in recipient than in control fish at all sampling times, except at 40 days p.e., though no statistically significant differences were observed. Serum peroxidases were higher in all recipient fish than in control ones, with the highest stimulation index at 40 days p.e. Within recipient fish, no differences were detected between sampling times in any of the measured activities. The possible implication of these immune factors in the high susceptibility of D. puntazzo to this enteromyxosis is discussed.

An unidentified epi-epithelial myxosporean in the intestine of gilthead sea bream Sparus aurata L.

In the course of experimental infections of gilthead sea bream Sparus aurata with the myxozoan Enteromyxum leei, stages of an unidentified myxozoan were observed attached to the intestinal brush border of some fish. Infection levels of the parasite, which was named "epi-epithelial myxosporean" (EEM) were recorded, and its structure was studied by light microscopy (LM) and electron microscopy (EM). In situ hybridisation (ISH) probes specific for E. leei were developed and used to differentiate between the two parasites. The EEM parasite was observed only in epi-epithelial position on the intestine mucosa and never in any of the other tissues studied (kidney and gall bladder). Prevalence was variable, with values reaching 40.2%. With transmission EM, trophozoites displayed pseudopodia-like projections inserted in between the enterocyte microvilli, producing an intimate interface. No mucosal histopathology that could be attributed to the myxozoan was found. EEM stages did not stain with the E. leei-specific ISH probes. From the results of the LM, EM and ISH studies, we conclude that the EEM parasite found in gilthead sea bream intestine in both Mediterranean and Red Sea sites is a coelozoic myxosporean, distinct from E. leei.

Pathology of Edwardsiella tarda infection in turbot, Scophthalmus maximus (L.).

Macroscopic and histopathological changes in cultured turbot, Scophthalmus maximus (L.), in Spain caused by infection with Edwardsiella tarda are described. Eye tumefaction, inflammation, haemorrhages, ascites and the presence of a purulent fluid were the main macroscopic lesions observed. Histopathological lesions were found in the kidney, spleen and liver. In the kidney and spleen these were characterized by a severe apostematous inflammatory reaction, with a large number of abscesses. The liver was affected to a lesser degree and only some phagocytes loaded with bacteria were observed. Ultrastructural observations indicated that macrophages were the main cell type implicated in the inflammatory response. Most of the bacteria observed within the phagocyte cytoplasm showed no degenerative changes and some were dividing. Degenerative changes observed in macrophages indicate their failure in preventing the infection.

Fractura avulsión aislada del troquín del húmero: a propósito de dos casos / Isolated avulsion fracture of the lesser tuberosity of the humerus: two cases

Objetivo. Estudio de dos casos de fractura-avulsión aislada del troquín del húmero tratados quirúrgicamente mediante la reinserción anatómica del mismo utilizando anclajes óseos.Material y método. Se describen dos casos de esta infrecuente lesión y los estudios de imagen realizados para su diagnóstico preciso. Se trata de una fractura-avulsión, que puede pasar desapercibida en las radiografías iniciales, siendo diagnosticada de forma tardía ante la persistencia de una disfunción dolorosa del hombro afecto y la realización de pruebas de imagen complementarias. Resultados. En ambos casos los pacientes lograron una recuperación funcional completa del hombro. Conclusiones. El tratamiento quirúrgico es preferible en los casos diagnosticados precozmente, y debe incluir la exploración de posibles lesiones asociadas de la porción larga del bíceps y del manguito de los rotadores, así como su reparación anatómica

Purpose. A study of two cases of isolated avulsion fracture of the lesser tuberosity of the humerus treated surgically by anatomical reattachment using bony anchors is reported. Material and methods. Two cases of this rare lesion are described as well as the images obtained for its precise diagnosis. The condition is an avulsion fracture, which is apt to be overlooked in initial X-rays, and only diagnosed when the persistence of a painful dysfunction of the affected shoulder leads to further imaging. Results. In both cases reported the patients achieved a complete functional recovery of their shoulders. Conclusions. In cases that are diagnosed early the treatment of choice is surgery. It must include the examination of the long portion of the biceps and the rotator cuff, to determine the presence of possible lesions and carry out their anatomical repair

Epidemiology of Cryptosporidium molnari in Spanish gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) cultures: from hatchery to market size.

A long-term epidemiological study of Cryptosporidium molnari in aquacultured European sea bass (ESB) and gilthead sea bream (GSB) was performed in different types of facilities on the Atlantic, Cantabric, and Mediterranean coasts. Four types of studies were carried out. In study A, fish raised from juveniles to marketable size (ongrowing stage) were periodically sampled in three different types of cultures. Studies B and C focused on hatchery and nursery facilities. In study D, occasional samplings were performed during mortality or morbidity outbreaks. As a general trend, C. molnari was more prevalent in GSB than in ESB. Data on the distribution pattern of C. molnari in total sampled GSB (studies A, B, and D) had a variance higher than the mean (overdispersion). In GSB (study A), the type of ongrowing system (sea cages, earth ponds, or indoor tanks) was found to have no significant effect. There was a significant relationship between the presence of the parasite and both fish weight and season. The highest infection values were recorded in spring. Prevalence and intensity had convex weight profiles, with a peak in 30- to 100-g fish. In study D, the prevalence of infection was higher in fish recently introduced in sea cages and in preongrowing systems. In studies B and C, fish were almost never infected before entering the postlarval and nursery facilities. The parasite seems to enter the host mainly through the water in production steps with less stringent water treatment. Recirculation systems and fish cannibalism could contribute to oocyst concentration and dispersion in aquaculture facilities.

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.

Histopathology of cultured sea bream Sparus aurata infected with sanguinicolid trematodes.

The present study is the first report of a sanguinicolid infection affecting sea bream Sparus aurata cultured in net cages in the NE of Spain. The disease was associated with trickling mortalities during the cold season (1999 and 2000). Examination of gill wet mounts of the affected population revealed that sanguinicolid infection was present in 82.6 and 100% of the fish sampled in 1999 and 2000, respectively. Adult flukes, which were located in the kidney, were tentatively identified as members of the family Sanguinicolidae, subfamily Cardicolinae. Eggs and miracidia were found in the gill vascular structures. The inflammatory response triggered by the parasites was moderate and the lesions caused by either eggs and miracidia in the gills or adult flukes in the kidney were not extremely severe, possibly because of the moderate intensity of the parasitosis. Histological observations of sanguinicolid infected sea bream presented here are compared with those reported in other fish species. The role played on sea bream morbility and mortality by other factors (occurrence of a simultaneous moderate monogenean infection, immunological impairement related to low water temperatures) is discussed.