The effects of endotoxin on b-adrenergic-mediated relaxation were investigated in the equine digital artery (EDA). Possible involvement of cyclooxygenase-2 (COX-2) in endotoxin-induced effects and basal EDA b-adrenoceptor functionality was also evaluated. Endothelium-intact (e+) and/or -denuded (e) EDA rings were incubated overnight with lipopolysaccharide (LPS), LPS + NS398 (selective COX-2 inhibitor) or NS398 alone. Vessel rings were then mounted in organ baths and relaxant responses to isoproterenol (ISOP) recorded on U44069-induced pre-contraction. Response to ISOP was further evaluated in either incubated or freshly isolated (e) rings acutely exposed to NS398. Fresh and incubated (e) EDAs were also analysed for COX-2 expression by Western blotting. LPS caused endothelium-dependent enhancement of b-adrenergic mediated relaxation. NS398 did not reverse endotoxin effects, suggesting that COX-2 did not have a mediating role. In the absence of LPS, NS398 significantly increased ISOP-induced relaxation. This finding, together with immunoblot detection of COX-2 in both fresh and incubated (e) vessels, revealed the existence of a constitutive COX-2 exerting tonic inhibitory modulation on EDA b-adrenergic-mediated relaxation. The results support the possible role of endotoxin in the vascular disturbances associated with equine laminitis. Moreover, the involvement of COX-2 in the physiological regulation of EDA tone warrants further clinical investigation into the efficacy and safety of selective COX-2 inhibitors on digital circulation in horses.

The development of primary cultures and cell lines from aquatic organisms is a valuable tool for a wide range of research activities applied to aquaculture. Despite several efforts, derivation and long-term culturing of primary hepatocytes from marine vertebrates are still rare and unsuccessful. This is the first report to fully characterize long-term cultures of primary hepatocytes from the European seabream, Sparus aurata L. (Osteichthyes, Sparidae) (SaHePs). In this new model, hepatocyte cells were long-term viable, active proliferating, and fully retained liver function up to 3 weeks. SaHePs expressed a differentiated phenotype, owing to the reacquisition of the peculiar cytoarchitecture with the complete assembly of cytoskeletal and junctional network, as shown by the production and immunolocalization of several polarity markers and cytoskeletal proteins (MDR1, ZO-2, C-CAM1, Vimentin, Cadherin, beta-Tubulin, beta-Catenin, beta-Actin). Cytostructural analysis to identify polarized expression and bile canaliculi formation was performed by immunofluorescence and contrast phase microscopy. Long cultured SaHePs also demonstrated evidence of Albumin, alpha 1-Antitrypsin (AAT) and alpha-Fetoprotein (AFP) synthesis, expression of the detoxifying metabolic enzyme cytochrome P-4501A (CYP 1A), and production of hepatocyte specific cytoskeleton proteins, such as Cytokeratin 8 (CK8) and Cytokeratin 18 (CK 18). The presence of specific markers for hepatic phenotype, detected by immunocytochemistry and Western blot analysis, is suggestive of the full maintenance of a highly differentiated phenotype and hepatic maturation. These data demonstrate that SaHePs can be long cultured without losing the hepatic functionality. This study provides a useful tool for innovative research applications in fish toxicological, pathological, and physiological studies, as one of the few hepatic, functionally active, in vitro model from marine fish.

Hepatozoonosis caused by Hepatozoon canis (Eucoccidiorida, Hepatozoidae) is among the most widespread vector-borne infections of dogs, primarily transmitted by Rhipicephalus sanguineus sensu lato ticks. Based on the absence of a consensus on the treatment regimes for canine hepatozoonosis, the present study aimed to evaluate the efficacy of imidocarb dipropionate (5-6 mg/kg subcutaneously once a week for 6 weeks), and of toltrazuril/emodepside (Procox(®), 15 mg/kg once a day for 6 days) in association with clindamycin (15 mg/kg once a day for 21 days) in treating naturally infected dogs. At the enrollment time (T0), 32 dogs, cytologically or molecularly positive for H. canis, were assigned to test and control groups. Animals were treated according to the specific therapeutic protocol, and the presence of H. canis gamonts was assessed weekly by cytology and PCR throughout six months (T1-T19). In addition, any abnormality in leucocyte morphology was evaluated and recorded. Results indicate that, in spite of a reduction in the percentage of infected dogs, both treatments did not provide parasitological cure. Accordingly, new treatment protocols or active compounds against H. canis should be investigated.

During the period October 2010 - October 2011 the WWF Rescue Centre of Molfetta (Italy) referred 134 loggerhead sea turtles (Caretta caretta) to the Faculty of Veterinary Medicine of Bari (Italy) for clinical evaluation following to bycatch in bottom trawling in Manfredonia Gulf (Southern Adriatic Sea, Italy). After biometric assessment, all the turtles underwent clinical evaluation and radiographic examination to check for lesions ascribable to the capture (carapace and plastron trauma, skull or limb fractures, pneumonia subsequent to forced submergence and drowning). Dorsoventral, lateral and craniocaudal (horizontal beams) projections of head, total body and limbs were performed. In 8 turtles (6%) the presence of fish hooks in upper gastrointestinal tract was assessed: 3 animals presented an evident fishing line, while the presence of the hooks resulted accidentally at the x-ray examination without apparent clinical abnormalities in the other subjects. 4 hooks were localized in the intracoelomatic tract of the esophagus, in correspondence of tracheal bifurcation, and the other ones in cervical esophagus. In one case the hook was manually removed after sedation, while the others required surgical procedures. The hooks located in upper esophageal tract were removed after cervical esophagotomy, while the removal of the ones positioned deeper in non papillated esophagus was achieved after a transversal supraplastron surgical approach to access the coelomic portion of the esophagus. During surgery, one of the turtles, presented in critical conditions, revealed a long fishing line attached to the hook, so a right prefemoral approach to coelomic cavity was performed in order to remove it from intestinal lumen with multiple enterotomies on the exteriorized small intestine. In 3 turtles the hook had pierced the esophageal wall and were located extra-lumen, giving rise to large inflammatory responses with granulomatous abscessations, which caused the displacement of trachea and the partial obstruction of esophageal lumen. Nonetheless, surgery proved solving, and adequate post-surgical management (with 2 to 5 weeks of hospitalization) allowed the total recovery and the release of the turtles. Data from this study show that the major risks of longline fishing bycatch are not only the presence of tracts of lines in the gastrointestinal tube, as reported by numerous Authors, but also the hook itself, as it can give rise to foreign body granulomatous reactions that can occlude the gastrointestinal tract, and impair the feeding capacity of the turtles.

The large majority of studies on the genotoxic hazard of PAHs polluted water widely applied the ENA assay as versatile tool in large number of wild and farmed aquatic species. Nuclear abnormalities are commonly considered to be a direct consequence of genotoxic lesions in DNA macromolecule, and such evaluation might be helpful in identifying the genotoxic damage induced by the most harmful PAHs such as B[a]P. Regarding at the fish species subjected to aquaculture, most of the toxicological data come from wild fish and mainly focus on freshwater fish, but very little is known for other marine major aquacultured species. The gilthead sea bream (Sparus aurata L.) is the most economically important sparid species cultured along the Mediterranean costs, and it has been proved a very sensitive species to acute B[a]P exposure. However, further investigation is needed on several other types of genotoxic assessments, especially for chronic effects. This work was totally based on an in vitro model for chronic toxicity, using long-term S. aurata hepatocytes in primary culture, continuously exposed to low levels of BaP, over a prolonged period of time, to provide evidences for latent toxicity response. We aimed to investigate the kind of nuclear damage in gilthead sea bream hepatocytes continuously exposed to B[a]P sublethal doses. Cells were exposed to several B[a]P concentrations (10 μg/mL, 1 μg/mL, 1 ng/mL, 1 pg/mL) for two exposure times (24 and 72 h), and then tested both for apoptosis induction and for nuclear abnormalities by immunofluorescence analysis. The presence of severe nuclear damage, revealed cells progressing towards abnormal genotypes, due to a series of aberrant mitosis followed by unequal distribution of chromosomal content. The nuclear atypia (NA) more frequently observed were: a) micronuclei (MN); b) nuclear buds or blebs (NBUDs); c) notched nuclei; d) lobed nuclei; e) nuclei with nucleoplasmic bridge (NPBs); f) nuclei squashed, with a residual nuclear membrane; g) open nuclei, with membrane tape unrolled; and h) apoptotic bodies. Our results showed at medium-low doses a sustained genotoxic response, whose potency increased with the exposure time, becoming apparent as apoptosis induction, both by cell surface and nuclear changes. At the lowest doses, the longer was B[a]P exposure, greater was the involvement on masses of replicating cells, establishing the connection between the escape from apoptosis and the selection of tumoral cell evolution. In view of these results, there is no evidence of a threshold dose below which B[a]P was found not to be genotoxic in sea bream cultured hepatocytes.

Benzo[a]pyrene (B[a]P) is the most studied dangerous polycyclic aromatic hydrocarbon for its hepatotoxic, carcinogenic, mutagenic, teratogenic, and immunosuppressant effects, which can affect both wild and farmed marine fish through the trophic chain. This study investigated, for the first time, the chronic effects induced in vitro by B[a]P prolonged exposure on gilthead sea bream (Sparus aurata L.) hepatocytes, evaluating the cellular and nuclear latent damage. The purpose was to characterize the kind of B[a]P cyto- and genotoxic damage by morphological and immunocytochemical parameters applied in combination with the use of multiple assay endpoints. In light of our results, the short-term effects at higher B[a]P doses were linked to higher cytotoxicities and necrotic lysis, whereas a sustained inflammatory response at medium-low doses was perceived as a mitochondria-mediated apoptosis, both by surface and nuclear morphological changes. The strong immunoreactivity for the cleaved caspase-3 showed that the labeled cells committed suicide by apoptosis. B[a]P involvement on carcinogenesis comes from prolonged exposure at lower doses, establishing the connection between the escape from apoptosis and the selection of a tumoral phenotype. Cells colabeled with proliferating cell nuclear antigen/caspase-3 within the proliferative foci, were proliferating transformed oval stem cells, which escaped the suicide by apoptosis allowing cancer development. Finally, it was established that sea bream cultured hepatocytes are highly sensitive to chronic B[a]P exposure, as serious genotoxic effects were found even at the lowest doses.

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Canine parvovirus (CPV) modified live virus vaccines are able to infect vaccinated dogs replicating in the bloodstream and enteric mucosa. However, the exact duration and extent of CPV vaccine-induced viremia and fecal shedding arenot known. With the aim to fill this gap, 26 dogs were administeredtwo commercial vaccines containing a CPV-2 orCPV-2b strain and monitored for 28 days after vaccination. By using real-timePCR, vaccine-induced viremia and shedding were found to be long lasting for both vaccinal strains (19-22 and 12-19 mean days, respectively), occurring at higher loads for the CPV-2b strain. With neither vaccine there was any interference with in-clinic or hemagglutination testing. The present study adds new insights into the CPV vaccine persistence in the organism and possible interference with diagnostic tests.

Neural Progenitor Cells (NPCs) have gathered more and more attention in the field of Neural Stem Cells (NSCs). However, the multilineage differentiating behavior of these cells and their contribution to tissue regeneration, almost in lower vertebrate taxa, remain unknown. Since the early 1970s, many comparative studies have been performed using immunocytochemical screening on the brains of several vertebrate taxa, including teleosts, in order to identify these cells, even if the data are sometimes contrasting. This study aims: (1) to investigate in vitro the potential proliferative role of NPCs and Radial Glia Progenitors (RGP) in seabream neurogenesis; (2) to reveal the strict ability of fish NSCs to undertake the multilineage development and differentiation in neurons, astrocytes and oligodendrocytes. By the use of double Immunofluorescence (IF) analysis and phase contrast microscopy, we identified the multilineage differentiation and the exact cell morphology. We demonstrated that NSC can self-renew and differentiate into different types of neurons or glial cells during extended culturing. Mature neurons expressed specific neuronal markers; they could differentiate during long term culturing, generating an extensive neurite growth. Glia was found highly mitotic and could developed mature astrocytes and oligodendrocytes. Glial cells were assessed by Glial Fibrillary Acidic Protein (GFAP) reactivity; neurons and myelinating oligodendrocytes were immunostained with cell-specific markers. This work provide that the multilineage differentiation potential of seabream neural cell progenitors might be a useful tool for neurodegenerative diseases, being a promising approach for repairing the CNS injuries, also in other animals, as a new coming strategy for function recovery of damaged nerves.

Data on reptile analgesia are scarce for nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids and almost completely lacking in sea turtles, even though emergencies requiring correct pain management are very frequent in their rehabilitative medicine; therefore, dosage regimens extrapolated from other species involve the risk of clinical failure and damage to the animals. We describe the pharmacokinetic behavior of meloxicam in the loggerhead sea turtle (Caretta caretta). We chose meloxicam because of its selective anticyclooxygenase- 2 activity and lesser adverse side effects. No data are available on the capacity of turtles to tolerate NSAIDs, so we chose a dose of 0.1 mg/kg of meloxicam. Plasma concentrations of meloxicam were unexpectedly low both for intravenous (IV; maximum concentration [Cmax]=0.04±0.02 µg/mL) and intramuscular (IM; Cmax =0.07±0.09 µg/mL) administration. A double-peak phenomenon occurred after both IV (time for second peak concentration Tmax2 = 10.33±10.89 h) and IM (Tmax2=1.17±0.75 h). The second peak after IM injection was premature, so some difficulty and delay in absorption appears to be an appropriate explanation. Furthermore, the area under the curve, and therefore systemic bioavailability (F531.82±28.24%), after both IV (0.30±0.29) and IM (0.10±0.03) injection appeared particularly limited. Terminal elimination slope and mean residence time indicated fast elimination after IM dosing; as a consequence, plasma concentrations dropped below analytic limits in 8 h. Considering that IM is the favored route of administration of drugs in rescue centers, it is unlikely that meloxicam at 0.1 mg/kg is an appropriate choice, particularly in long-term pain management protocols

We report the surgical techniques used to remove accidentally ingested hooks and branchlines localized in different parts of the digestive tract of 129 loggerhead sea turtles Caretta caretta, together with the characteristics and localization of lesions, and final outcome related to their severity. Hooks were removed from the cervical esophagus via the ventral surface of the neck, while the supraplastron approach was performed for hooks wedged in the intracoelomic portion of the esophagus. An approach through the left axillary region was preferred for fishhooks in the stomach, while hooks and long branchlines in the intestine or pyloric area were removed by approaching the coelomic cavity through the right or left prefemoral fossa. The ingestion of fishhooks, and/or longlines, often induces severe injuries in the digestive tract that could lead to the death of the turtles, with the extent of damage engendered by lines often more severe than that caused by hooks, leading to strangulation, intussusception, and tears that require resection of long tracts of intestine. Spontaneous expulsion of hooks, even where possible, involves long waiting times, with the possible impairment of the turtle's clinical condition, and should be avoided when the line is evident or suspected. The development of diversified surgical techniques enabled us to approach the coelomic cavity with minimally invasive and easy-to-perform methods, and survival rates proved very satisfactory.

Severely debilitated or post-surgical sea turtles often suffer from anorexia, making their management very challenging. In cases like these, a nutritional support is mandatory. Common practice in rescue centres is assisted feeding, daily administered via a soft tube passed by mouth to stomach. This procedure is relatively easy in most reptile species, but it results very difficult in chelonians, and particularly in large sea turtles, as access to mouth and oesophagus can become impossible if the animal withdraw the head. Furthermore, this practice can result a source of considerable stress in wild animals, and in sea turtles it becomes very messy because of the particularly narrow gastroesophageal sphincter. The placement of a permanent oesophagostomy tube can considerably simplify the daily administration of assisted nutrition. Drugs (antibiotics, vitamins, etc.) and fluids, essential in the clinical management, are as well easily administered through the permanent oesophagostomy. This procedure was performed in 5 severely ill Loggerhead sea turtles (Caretta caretta) in order to assure a correct assisted feeding. All patients were anaesthetized with 4-5 mg/kg intravenous propofol. Before placement, the tube length was pre-measured from the lateral side of the neck to the left pectoral scute, then permanently marked. With the extended neck, a curved hemostat was introduced through the mouth into the esophagus and laterally displaced. This caused the skin to tent and the carotid and jugular veins to slip dorsally or ventrally to avoid them to be incised. A small incision was made trough the skin and the wall of the esophagus with a scalpel blade at the tip of the hemostat, that has been forced outside by blunt. The incision has to be as caudal as possible in the neck to avoid the turtle to entangle a limb and extract the tube. The tip of the tube was grasped with the clamp, pulled through the incision and withdrawn trough the mouth to the marked point. Then, the tube was redirected into the oesophagus and pushed up to the stomach. Levin’s tubes 4-5 mm diameter with radiopaque guide were used, to verify the correct placement by x-ray examination. After placement the tube was sutured to the skin just next to the incision with nonabsorbable sutures; the extended length of the tube was secured to the edge of the nucal scute with a suture and to the carapace with cyanoacrylic glue. Broad spectrum antibiotics were administered after the surgical procedure. Patients were fed daily through the tube with homogenized fish and shellfish, supplemented with vitamins. After food administration, the tube was washed with few millilitres of saline solution to avoid its obstruction. The tube was well tolerated, and the turtles were able to eat normally in 2-3 weeks while it was still in place. The tube was kept in place for two more weeks after appetite had returned to normal. If the tube has to be held in place for several weeks, it is possible that reparative reactions expel stitches and the tube needs to be sutured again.