Use of Rapid Cytochemical Staining to Characterize fish blood granulocytes in Species of Special Concern and Determine Potential for Function Testing

Studies of innate immunity in fish species of special concern are essential for better understanding of their health status during hatchery rearing conditions. The cytochemical and morphological characterizations of blood granulocytes have been used to provide information about phylogenetic differences and determine the potential use of neutrophil function assays. Rapid, simple, cytochemical staining kits used routinely for staining mammalian granulocytes have been used to characterize granulocytes from blood of four fish species: Arctic grayling, cutthroat trout, June sucker, and shovelnose sturgeon. Blood smears were stained with Peroxidase 391 (myeloperoxidase, MPO), alkaline phosphatase (AP), Periodic Acid Schiff (PAS) and Diff-quick stain; examined using bright field and differential interference contrast microscopy. Granulocytes on blood smears were evaluated based on the cell morphology, and presence or absence of the specific chromogen. Presence of lymphocytes, monocytes, platelets/thrombocytes and granulocytes was determined in all fish species. Arctic grayling, June sucker, and cutthroat trout had MPO positive granulocytes, while shovelnose sturgeon heterophils had positive reaction for leukocyte AP, but not MPO. Presence of MPO indicated potential to measure oxidative burst and degranulation of neutrophil primary granules in Arctic grayling, cutthroat trout and June sucker. Absence of MPO in shovelnose sturgeon suggested use of different enzyme marker (AP) in degranulation assay for this species. Standardization of cytochemical techniques allowed forrapid screening of leukocyte types, reducing the number of fish, time and effort to select adequate neutrophil function assays to be used in studies of health status in species of special concern

Mapping the teaching of aquatic animal veterinary medicine in the European Union and European Free Trade Area

Aquatic animal production is the fastest growing food sector globally. Aquaculture and fisheries are very dynamic sectors in the EU and the number of ornamental aquarium pets is increasing. Veterinarians have a fundamental role to play by ensuring health and welfare of aquatic species, productivity and profitability of fish farming, public health and ecosystem conservation. This study investigates how the undergraduate curriculum prepares future veterinarians for such roles by analysing data from the 77 European veterinary education establishments based in EU and the European Free Trade Area. Over 95 per cent of these establishments incorporate teaching in aquatic animal veterinary medicine in their curriculum, while the great majority do so within the core curriculum. Almost half of the establishments provide teaching in aquatic animal veterinary medicine as separate subjects. Many establishments (>40 per cent) provide such training as elective option in their undergraduate curricula or as postgraduate opportunities to enhance Day One Competences. The veterinary education establishments integrating adequately aquatic animal veterinary medicine in their curriculum are evenly distributed in all regions of Europe. Veterinarians are trained and empowered by legislation to assess health of aquatic animals, to diagnose, to prescribe medicines, to notify for diseases and to ensure safe food for the consumers. Veterinary education establishments should encourage training of veterinarians to follow a career in aquatic animal veterinary medicine

Autogenous vaccines in aquaculture: tool to combat resistance of bacteria to antibiotics?

New technological progress and increased demands for fish as a source of animal protein are driving significant growth of aquaculture production. Intensification of production increases the severity and frequency of infectious disease outbreaks, and so requires significant effort to prevent and control disease. Because of the global crisis of bacterial resistance to antibiotics, the use of antibiotics in aquaculture is increasingly subjected to strict control and regulatory measures, leading to potential misuse. The lack of availability of approved veterinary medical products for use in aquaculture, combined with the risk of drug resistance development and antibiotic residues in fish flesh or water, support the development of preventive actions, including vaccines. However, the diversity of species and aquaculture production methods, including epidemiological units and their links, results in economic challenges for commercial vaccine development and authorization. As a possible response to the increasing demand for less antibiotic use in fish farms, and to the expenses associated with novel veterinary product development, there is a need for increased use of safe and effective autogenous vaccines in aquaculture. Regulatory processes for autogenous vaccine production, approval and application should recognize the specificities of epidemiological units and their links in aquatic animal production facilities. The joint efforts of regulatory authorities, producers, and veterinary services to follow veterinary biosecurity principles, including risk analysis, surveillance, and selection/prioritization of pathogens, are essential to provide maximum safety and efficacy of autogenous vaccines as disease prevention and control tools within larger areas, such as compartments and zones, and allow for reductions in antibiotic use

Assessment of heavy metal load in chub liver (Cyprinidae – Leuciscus cephalus) from the Nišava River (Serbia)

The Nišava River plays an important role as the source for both drinking water and agricultural irrigation dueto its hydrological and geomorphological characteristics as the largest river in the region of southeast Serbia.In this study we used the liver of the chub (Leiciscus cephalus) as a tool for biomonitoring heavy metalaccumulation along the river. Chub specimens were sampled from two localities (one at the border withBulgaria and a second in the city of Niš). Concentrations were estimated for six heavy metals (iron,cadmium, copper, zinc, lead and manganese) in chub liver. Low bioconcentration level was observed formost of the metals and the concentrations corresponded to the nominal concentration in livers of fishinhabiting metal unpolluted streams and rivers. However, cadmium concentration in the chub liver exceeded0.5 mg kg-1, a several hundred folds increase from nominal concentration indicating a potential toxicexposure of the fish and of the stream ecosystem to this heavy metal. Hepatosomatic indices were calculatedand tested for the impact of metal concentrations on liver size. A decrease of the hepatic index was observedin fish with higher cadmium concentration, suggesting a possible impact on the health of the chub populationin the Nišava River

Histopathology of fathead minnow (Pimephales promelas) exposed to hydroxylated fullerenes

Hydroxylated fullerenes are reported to be very strong antioxidants, acting to quench reactive oxygen species, thus having strong potential for important and widespread applications in innovative therapies for a variety of disease processes. However, their potential for toxicological side effects is still largely controversial and unknown. Effects of hydroxylated fullerenes C60(OH)24 on the fathead minnow (Pimephales promelas) were investigated microscopically after a 72-hour (acute) exposure by intraperitoneal injection of 20 ppm of hydroxylated fullerenes per gram of body mass. Cumulative, semi-quantitative histopathologic evaluation of brain, liver, anterior kidney, posterior kidney, skin, coelom, gills and the vestibuloauditory system revealed significant differences between control and hydroxylated fullerene-treated fish. Fullerene-treated fish had much higher cumulative histopathology scores. Histopathologic changes included loss of cellularity in the interstitium of the kidney, a primary site of haematopoiesis in fish, and loss of intracytoplasmic glycogen in liver. In the coelom, variable numbers of leukocytes, including many macrophages and fewer heterophils and rodlet cells, were admixed with the nanomaterial. These findings raise concern about in vivo administration of hydroxylated fullerenes in experimental drugs and procedures in human medicine, and should be investigated in more detail.This article is published as Jovanović, Boris, Elizabeth M. Whitley, and Dušan Palić. "Histopathology of fathead minnow (Pimephales promelas) exposed to hydroxylated fullerenes." Nanotoxicology 8, no. 7 (2014): 755-763. doi: 10.3109/17435390.2013.828794.</p

Anesthetic Efficacy of Tricaine Methanesulfonate, Metomidate and Eugenol: Effects on Plasma Cortisol Concentration and Neutrophil Function in Fathead Minnows (Pimephales promelas Rafinesque, 1820)

Anesthetic efficacy, plasma cortisol concentration, and two parameters of neutrophil function (oxidative burst and degranulation of primary granules) were compared among three anesthetics in the fathead minnow: tricaine methanesulfonate (MS 222), metomidate hydrochloride (MTMD), and eugenol (EUG). The optimum anesthetic concentration was determined as: MS 222 75 mg L− 1, EUG 30 mg L− 1 and MTMD 4 mg L− 1. Handling and crowding stress was induced in fish with (SA) and without (S) anesthetic. Plasma cortisol concentration was measured at 0, 30, 90, and 240 min after stress and found to increase at 30 min post-stress in S and SA MS 222 groups, but not in SA MTMD and SA EUG groups. To test the effects of different anesthetics on neutrophil function, fish were divided into a baseline control group, a group exposed to handling and crowding stress (S) and a stressed anesthetized group (SA). Fish were assayed for neutrophil function before and after stress (24 h, 72 h and 7 days). The degranulation of neutrophil primary granules was measured as exocytosis of myeloperoxidase (MPO) using 3, 3′, 5, 5′-tetramethylbenzidine as a substrate. Degranulation of primary granules was decreased to 60–75% of non-stressed control in stressed and fish treated with MS 222, and was not affected when MTMD and EUG were used. The degranulation of primary granules proved to be a useful assay for measuring the effects of stress on neutrophil function in fish. Eugenol and metomidate prevented stress-induced decrease of neutrophil function while MS 222 did not

Immunomodulatory Effects of beta-Glucan on Neutrophil Function in Fathead Minnows (Pimephales Promelas Rafinesque, 1820)

Stimulatory effects of yeast b-1,3–1,6-glucans on neutrophils have long been recognized, but effects of glucans on degranulation of primary granules in fish neutrophils have not been previously reported. Neutrophil function was monitored during in vitro and in vivo application of glucans to non- (NS), acute- (AS) and chronically stressed (CS) fish. b-Glucan proved to be a strong and quick (80%, 2 min) stimulant of degranulation. Dietary glucan increased degranulation in NS fish, and prevented a decrease in AS fish. Degranulation in CS fish returned to NS levels 3 days after the glucan diet was fed. Fathead minnows appear to be a useful model to investigate neutrophil degranulation in fish exposed to different environmental conditions and immunomodulators. Use of b-glucans in fish diets prior to AS and during chronic stress can enhance neutrophil function, potentially increasing disease resistance and survival rates after transportation or exposure to poor water quality

Fish Assemblages of the Upper Little Sioux River Basin, Iowa, USA: Relationships with Stream Size and Comparison with Historical Assemblages

We characterized the fish assemblages in second to fifth order streams of the upper Little Sioux River basin in northwest Iowa, USA and compared our results with historical surveys. The fish assemblage consisted of over twenty species, was dominated numerically by creek chub, sand shiner, central stoneroller and other cyprinids, and was dominated in biomass by common carp. Most of the species and the great majority of all individuals present were at least moderately tolerant to environmental degradation, and biotic integrity at most sites was characterized as fair. Biotic integrity declined with increasing stream size, and degraded habitat in larger streams is a possible cause. No significant changes in species richness or the relative distribution of species\u27 tolerance appear to have occurred since the 1930s