Preliminary morphometrics of spleen and kidney macrophage aggregates in clinically normal blue gourami Trichogaster trichopterus and freshwater angelfish Pterophyllum scalare

Macrophage aggregate (MA) morphometry and pigment composition are believed to be dependent on the species, age, and health status of the fish. The aim of this study was to characterize a "normal" morphometry baseline of spleen and kidney MAs in blue gourami Trichogaster trichopterus and freshwater angelfish Pterophyllum scalare. Three size-classes of clinically normal fish were analyzed. Blue gourami and freshwater angelfish were obtained from three local ornamental fish farms; for each size-class, 10 fish from each farm were analyzed. Hematoxylin- and eosin-stained tissue sections were analyzed by light microscopy at 100x magnification and an image analysis program. The percentage of tissue occupied by MAs, MA size, and MA number were calculated on three arbitrarily selected fields of view from each spleen and kidney. In clinically normal blue gourami, increases in the percentage of tissue occupied by MAs and in MA size were associated with an increase in fish size, but in clinically normal angelfish no correlation was observed. Furthermore, in angelfish, a high variability in MA morphometry was observed, even among fish from the same sample group. In both species, a significant difference in the value of the morphometric parameters was observed among farms. Because iridoviruses inhibit macrophage activity and (possibly) proliferation, MAs in 25 clinical cases of iridovirus-infected blue gourami were analyzed. Preliminary data indicate that in iridovirus-infected blue gourami, there is a decrease in MA size and MA number compared with those of healthy fish.     

Isolation and Classification of a Nocardia species from Diseased Giant Gourami Osphronemus goramy

Abstract

An enzootic caused by bacteria occurred among cultured giant gourami in two freshwater fish farms in Kagoshima, Japan, in 1982. Typical external characteristics of the diseased fish were sloughed scales, exophthalmia, and opaque eyes. Internally, numerous creamy white nodules were found in the gills, spleen, kidney, and liver and a lesser number in the bladder and stomach. Nocardia sp. was isolated from spleen and kidney tissues of all diseased fish examined. To clarify the taxonomic position of the isolates, 164 tests were performed, and results were compared to reference strains of six species of Nocardia and one species of Rhodococcus. The isolates formed a homogenous group in a single taxon at a similarity of 98.7% based on an average linkage analysis and were clearly separated from the reference strains. The isolated strains were more similar to Nocardia kampachi than to the other representative species. However, 12 distinct differences were found between the isolates and N. kampachi, including growth on nutrient agar, sensitivity to 250 and 500 μg hydroxylamine/mL and 0.01% phenol, tolerance of 0.05% salicylate and 0.1% picric acid in brain-heart infusion medium, hydrolysis of starch, nicotinamidase reaction, and acid from fructose, sucrose, and dextrin. Accordingly, the isolates from the diseased giant gourami were classified as a new species of Nocardia.     

Communications: Isolation of Several Mycobacterium Species from Fish

Abstract

Several species of marine fish caught in the wild and of freshwater ornamental fish were used in this study. Infected organs (liver, spleen, and kidney) were sampled for mycobacteria. Decontaminated tissue samples were plated onto selective media for mycobacterial recovery. After initial isolation, fluorescent and acid-fast staining techniques identified bacterial colonies to genus. Profiles of biochemical growth characteristics were used to further identify the isolates to species. Five species of Mycobacterium were identified: M. simiae, M. scrofulaceum, M. marinum, M. chelonae, and M. fortuitum. Of these, M. simiae and M. scrofulaceum have not been previously reported from fish. Tissue samples containing focal granulomatous lesions were prepared for electron microscopic examination.     

Preliminary investigations of hydrogen peroxide treatment of selected ornamental fishes and efficacy against external bacteria and parasites in green swordtails

The objectives of these preliminary studies were to evaluate the use of hydrogen peroxide (H2O2) for the treatment of selected species of ornamental fishes and its efficacy in treating external bacteria and parasites. In the first part of the study, fish of five species (serpae tetra Hyphessobrycon eques (also known as Serpa tetra H. serpae), tiger barb Puntius tetrazona, blue gourami Trichogaster trichopterus, suckermouth catfish Hypostomus plecostomus, and green swordtail Xiphophorus hellerii) were exposed to H2O2 for 1 h at concentrations between 6 and 34 mg/L or for 24 h at concentrations between 1 and 6 mg/L. The results were species specific: green swordtails tolerated all of the treatments, serpae tetras and tiger barbs were sensitive only to the highest concentration, and mortalities of suckermouth catfish and blue gourami were recorded in every treatment. In the second part of the study, clinically healthy green swordtails and fish infested with external motile rod-shaped bacteria (i.e., Ichthyobodo spp., Trichodina spp., and Gyrodactylus spp.) were treated with several concentrations of H2O2. A single H2O2 treatment of 3.1 mg/L or more for 1 h effectively eliminated external bacteria, concentrations of 6.5 mg/L or more appeared to effectively kill Ichthyobodo spp., and none of the treatments tested was effective against Trichodina spp. or Gyrodactylus spp. These preliminary findings suggest that H2O2 is effective for treating certain external bacterial infections and flagellate infestations in some species of ornamental fish at the dosages tested. Other treatment regimens may need to be tested for effectiveness against Trichodina spp. and Dactylogyrus spp.     

Exposure to Perchlorate Induces the Formation of Macrophage Aggregates in the Trunk Kidney of Zebrafish and Mosquitofish

Abstract

Environmental contamination of ground and surface waters by perchlorate, derived from ammonium perchlorate (AP) and other perchlorate salts, is of increasing concern. Exposure to perchlorate can impair the thyroid endocrine system, which is thought to modulate renal and immune function in vertebrates. This study with zebrafish Danio rerio and eastern mosquitofish Gambusia holbrooki examined the histological effects of perchlorate on the trunk kidney, which in teleosts serves excretory and hemopoietic functions and therefore may be a target of perchlorate effects. Adult zebrafish of both sexes were exposed in the laboratory to waterborne, AP-derived perchlorate at measured concentrations of 18 mg/L for 8 weeks. Adult male mosquitofish were exposed to waterborne sodium perchlorate at measured perchlorate concentrations of 1–92 mg/L for 8 weeks. Control fish were kept in untreated water. The region of the body cavity containing the trunk kidney was processed from each fish for histological analysis. Macrophage aggregates (MAs), possible markers of contaminant exposure or immunotoxic effect, were present in the hemopoietic region of the kidney in both species exposed to perchlorate. The estimated percent area of kidney sections occupied by MAs was greater in zebrafish exposed to perchlorate at 18 mg/L (P < 0.05) than in controls. In male mosquitofish, the incidence of renal MAs increased proportionally with sodium perchlorate concentration and was significantly different from that of controls at 92 mg/L (P < 0.05). These observations confirm that in fish the kidney is affected by exposure to perchlorate. The concentrations of perchlorate at which the effects were noted are relatively high but within the range reported in some contaminated habitats.     

Effects of Metomidate Hydrochloride Sedation on Blood Glucose and Marketability of Transported Threespot Gourami Trichogaster trichopterus

Abstract

Our objectives were to determine whether sedation with metomidate hydrochloride (hereafter, “metomidate”) during transportation of threespot gourami Trichogaster trichopterus would prevent an increase in blood glucose levels and improve fish marketability (i.e., based on appearance and behavior) in comparison with unsedated controls. Threespot gourami are obligate air-breathers that possess a labyrinth organ, enabling the fish to respire air above the water surface; these fish should be lightly sedated during transport. Fish were transported for approximately 24 h via truck and domestic airline. Blood was sampled at 0, 2, 6, and 12 h posttransportation, and appearance and behavior were observed at 0, 2, 4, 6, and 12 h and 7 d posttransportation. Metomidate concentrations tested were 0.0 (control), 0.1, 0.2, 0.3, and 0.4 mg/L. At the concentrations tested, metomidate neither inhibited elevations in blood glucose nor improved marketability. Fish that were transported with 0.3-mg/L metomidate were less marketable based on behavioral indices, and fish that were transported with 0.4-mg/L metomidate had higher glucose levels than control fish. Use of metomidate as a transport sedative for threespot gourami should be considered with caution and may be problematic at the concentrations tested; however, further research examining additional indices of stress may clarify metomidate use for this species.

Received February 16, 2011; accepted December 18, 2011.     

Odontogenic hamartomas in cultured angelfish (Pterophyllum scalare)

BackgroundOdontogenic tumors present diverse clinical and histopathological behaviors that range from benign odontoma or hamartoma lesions to malignant tumors. In angelfish Pterophyllum scalare, findings of tumorous masses have been reported in the frontal region of the mouth, encompassing both the maxilla and mandible. Histologically, oral tumors have been characterized as angelfish lip fibroma or ameloblastoma.MethodsIn this report, we describe the occurrence of a compound odontoma/hamartoma in 60 angelfish from a population of 300, all of which were collected from a Mexican ornamental fish farm.ResultsAll fish presented a tumor-like mass on the rostral part of the mouth. Histological analyses revealed the tumor-like mass was covered by a hyperplastic stratified squamous epithelium, with fully differentiated denticles in various states of organization, immersed among normal dental tissues and showing a mild disorganized pattern. Radiograph analysis revealed radiopaque denticles in a radiolucent protuberance. Bacterial and viral isolation procedures and electron microscopy analysis revealed no presence of these microorganisms.Conclusion and clinical relevanceAlthough findings of odontogenic tumors in the mouth of angelfish are reported as sporadic, our study detected odontogenic mouth tumors in 60 angelfish, but the possible cause of alteration is not yet clarified. Further research is needed to clarify pathogenesis and to identify possible genetic abnormalities.     

Cytological comparison of fine-needle aspirates of liver and spleen of normal dogs and of dogs with cutaneous mast cell tumours and an ultrasonographically normal appearing liver and spleen

Staging of dogs with cutaneous mast cell tumours (MCTs) is an important diagnostic step. Aspiration of the liver and the spleen is often part of routine staging. This study cytologically compared mast cell numbers in fine‐needle aspirates of liver and spleen of clinically normal unaffected dogs with those of dogs with cutaneous MCT and an ultrasonographically normal appearing liver and spleen. The unaffected dogs (n = 32) were selected from humane society dogs, and the affected dogs (n = 51) were selected from hospital cases. There were no statistically significant differences in each of the parameters evaluated for the liver aspirates. For splenic aspirates, affected dogs showed significantly more mast cells per cluster (P = 0.04) and more isolated mast cells per slide (P = 0.03) compared with unaffected dogs. However, no clinically important difference existed between the unaffected and affected dogs; thus, routine aspiration of an ultrasonographically normal appearing liver and spleen of dogs with cutaneous MCT does not appear to be a clinically useful staging tool.     

Replacement of fish meal protein by surimi by‐product protein in the diet of blue gourami Trichogaster trichopterus fingerlings

Based on the nutrient requirement of Trichogaster trichopterus, a fish meal–based basal diet with 350 g/kg diet crude protein and 16.7 MJ/kg energy was formulated, in which the fish meal protein was replaced by surimi by‐product protein at 0.0 (control), 12.5, 25, 50, 75 and 100% levels. The formulated diets were fed ad libitum to T. trichopterus fingerlings (4.80 ± 0.03 g) in triplicate groups for 45 days in a closed water system. Eighteen fibre‐reinforced plastic tanks with 200 l of water were used for rearing the fish. Weight gain, specific growth rate, feed/gain ratio, protein efficiency ratio, nutrient retention and digestibility (protein and energy) of fish were not affected (p > 0.05) up to 50% fish meal protein replacement level by surimi by‐product protein. While whole‐body protein content of fish was marginally decreased, the lipid content was increased with increase in surumi by‐product incorporation level in the diet. The study results suggest that the fish meal protein, which is scarce and costly nowadays, could be replaced up to 50% by surimi by‐product protein in the diet of blue gourami without hampering the growth and nutrient utilization of fish.     

Prevalence of Infectious Pancreatic Necrosis Virus on Salmonid Fish Farms in Spain

Abstract

In Spain, salmonid fish farming was commercially developed in the 1960s, and now there are 140 private farms that depend heavily on imported embryonate eggs. Infectious pancreatic necrosis was first clinically diagnosed in Spain in 1970, but the virus (IPNV) was not isolated and identified until 1980. Since that time, researchers have isolated IPNV from other samples in Spain. A diagnostic survey was conducted to determine how prevalent IPNV is on fish farms in Spain and whether the virus has been responsible for some of the major financial losses occurring every year on these farms. In total, 236 samplings of rainbow trout Oncorhynchus mykiss from 31 farms in eight hydrographic areas were done over a 3-year period. Infectious pancreatic necrosis virus was isolated in 94 cases, and serotyping of the viral strains revealed that 81% of these isolates were strain Sp and 19% were strain Ab. Neither IPNV strain VR-299 nor rhabdovirus (as infectious hematopoietic necrosis virus or viral hemorrhagic septicemia virus) was detected in any of the samples.     

Parasite Infections in Danish Trout Farms

Samples from 5 Danish freshwater trout farms rearing rainbow trout (Oncorhynchus my kiss) were examined for parasite infections from October 1993 until November 1994 and recorded parasites are listed. In addition, results from an examination of a mariculture net cage system are presented as well. A total of 10 metazoan and 10 protozoan parasites were recorded. The metazoans included Gyrodactylus derjavini, Gyrodactylus salaris, Eubothrium crassum, Triaenophorus nodulosus, Proteocephalus sp., Diplostomum spathaceum, Tylodelphus clavata and Argulus foliaceus from the freshwater farms. The protozoans Hexamita salmonis, Ichthyobodo necator, Ichthyophthirius multifiliis, Apiosoma sp., Epistylis sp., Trichodina nigra, T. mutabilis, T. fultoni, Trichodinella epizootica, and an Ichthyophonus like intestinal parasite were also detected in the freshwater trout farms. Based on lectin binding studies, few fish were found positive for the myxosporean parasite PKX although no clinical cases were reported. In the mariculture system, Lepeophtheirus salmonis and Caligus elongatus were found.     

Evaluation of a point-of-care blood analyzer and determination of reference ranges for blood parameters in rockfish

OBJECTIVE: To compare values of blood parameters in rockfish obtained by use of a point-of-care portable blood analyzer with values determined by a veterinary diagnostic laboratory, calculate reference ranges for various blood parameters in black rockfish, and compare values of blood parameters in clinically normal fish with those of fish with clinical abnormalities. DESIGN: Prospective study. ANIMALS: 41 captive adult black rockfish (Sebastes melanops) and 4 captive adult blue rockfish (Sebastes mystinus). PROCEDURE: Rockfish were anesthetized with tricaine methanesulfonate for collection of blood samples. Heparinized blood samples were immediately analyzed with a point-of-care analyzer. Blood sodium, potassium, chloride, urea nitrogen, and glucose concentrations; Hct; pH; partial pressure of carbon dioxide; total carbon dioxide concentration; bicarbonate concentration; base excess; and hemoglobin concentration were determined. A microhematocrit technique was used to determine PCV, and a refractometer was used to estimate total plasma protein concentration. Paired heparinized blood samples were transported to a veterinary diagnostic laboratory for analyses. RESULTS: Data obtained with the point-of-care analyzer were reproducible; however, values for most blood parameters were significantly different from those obtained by the veterinary diagnostic laboratory. Fish with poor body condition had several blood parameter values that were lower than corresponding values in clinically normal fish. CONCLUSIONS AND CLINICAL RELEVANCE: Point-of-care blood analyses may prove useful in rockfish. Point-of-care data for a large number of clinically normal fish must be obtained for reference ranges to be calculated, and further assessments of clinically abnormal fish are necessary to determine the relevance of the data.     

Amoeba‐like Infections in Cultured Marine Fishes: Systemic Infection in Pompano Trachinotus falcatus L. from Singapore and Gill Disease Associated with Paramoeba sp. in Sea Bream Sparus aurata L. from Greece

Two cases of Amoeba‐like infections in cultured warmwater marine fish are described, an unusual systemic infection in pompano Trachinotus falcatus L. from Singapore and a gill infection in Mediterranean sea bream Sparus aurata L. All pompano showed marked systemic infection of Amoeba‐like parasites in gills, kidney, intestine, pancreas and spleen. The most severe lesions were in the gills and renal tissue with minimal tissue reaction in other organs.     

Detection of Infectious Pancreatic Necrosis Virus from the Leeches Hemiclepsis marginata and Hirudo medicinalis

Leeches have been reported to harbor several important fish pathogens, including spring viremia of carp virus, infectious hematopoietic necrosis virus (IHNV), and viral hemorrhagic septicemia virus (VHSV), and also may contain blood protozoa. In the present study, leeches were collected from water bodies located in Kurdistan province, Iran. The specimens were tested for IHNV, VHSV, and infectious pancreatic necrosis virus (IPNV) using the PCR method. The results showed that two different species of leeches, Hemiclepsis marginata and Hirudo medicinalis, were infected by IPNV among the seven species studied. The infected leeches were found in areas that were polluted with untreated sewage coming from upstream fish farms culturing Rainbow Trout Oncorhynchus mykiss. In addition, the fish at fish farms in the vicinity had been infected with IPNV 9 months previously. Our results showed that the virus causing infectious pancreatic necrosis is present in the leeches H. marginata and H. medicinalis, suggesting that leeches are a potential source of IPNV in fish farms.

Received October 14, 2015; accepted June 1, 2016 Published online September 29, 2016     

Effects of metomidate hydrochloride sedation on blood glucose and marketability of transported threespot gourami Trichogaster trichopterus

Our objectives were to determine whether sedation with metomidate hydrochloride (hereafter, "metomidate") during transportation of threespot gourami Trichogaster trichopterus would prevent an increase in blood glucose levels and improve fish marketability (i.e., based on appearance and behavior) in comparison with unsedated controls. Threespot gourami are obligate air-breathers that possess a labyrinth organ, enabling the fish to respire air above the water surface; these fish should be lightly sedated during transport. Fish were transported for approximately 24 h via truck and domestic airline. Blood was sampled at 0, 2, 6, and 12 h posttransport ation, and appearance and behavior were observed at 0, 2, 4, 6, and 12 h and 7 d posttransportation. Metomidate concentrations tested were 0.0 (control), 0.1, 0.2, 0.3, and 0.4 mg/L. At the concentrations tested, metomidate neither inhibited elevations in blood glucose nor improved marketability. Fish that were transported with 0.3-mg/L metomidate were less marketable based on behavioral indices, and fish that were transported with 0.4-mg/L metomidate had higher glucose levels than control fish. Use of metomidate as a transport sedative for threespot gourami should be considered with caution and may be problematic at the concentrations tested; however, further research examining additional indices of stress may clarify metomidate use for this species.     

Histology of some major immune system organs in Lake Van fish Alburnus tarichi (Güldenstädt, 1814) (Cyprinidae)

The teleostean immune system is variable between and within taxa in terms of morphology. Accordingly, in the current study, the histology of some immune organs (thymus, kidney and spleen) of Lake Van fish (Alburnus tarichi) was investigated. For this purpose, the tissues from eight mature fish were fixed and embedded in paraffin wax, and then the sections were stained with haematoxylin and eosin, Mallory’s triple, periodic acid-Schiff and Alcian blue (pH of 2.5) for histologic examinations. In the thymus, no clear discrimination of the cortex and medulla was observed. Lymphoid cells within a reticulo-epithelial network were the predominant cell type in the thymus. The other components in the thymus were macrophages and myoid, mast-like, plasma-like, cystic cells, Hassall’s corpuscles, and single or multiple epithelial cystic structures. In the kidney, the head kidney, a major haematopoietic site, consisted of lymphoid and non-lymphoid zones within a reticular network. Cells exhibiting mitotic figures were also detected in the haematopoietic tissue of the head kidney. Haematopoietic tissue was also found in the trunk kidney dispersed amongst the excretory components. The spleen was composed of red and white pulp. The red pulp comprised abundant erythrocytes, whilst the white pulp contained leucocytes with a reticular network. Ellipsoids were also determined in the white pulp. Melanomacrophage centres were found in all of the examined lymphoid tissues of the fish. These findings, which were reported, herein, for the first time will provide reference knowledge for future studies of this anadromous fish.     

Responses of Blue Catfish and Channel Catfish to Environmental Nitrite

Abstract

In nitrite-exposure experiments, percent methemoglobin, plasma nitrite concentration, and plasma chloride ion concentration were compared between channel catfish Ictalurus punctatus and blue catfish I. furcatus exposed to sublethal levels of nitrite for 48 h at 25°C. In nitrite-recovery experiments, fish exposed to elevated environmental nitrite for 12 h were transferred to freshwater, and blood characteristics were monitored during the 24-h recovery period. Blue catfish appeared to be more resistant to environmental nitrite than channel catfish. Methemoglobin levels (percent of hemoglobin in methemoglobin form) were significantly lower in blue catfish than in channel catfish. Maximum plasma nitrite concentrations were 137 mg NO₂-/L plasma in blue catfish and 164 mg NO₂-/L plasma in channel catfish. Percent methemoglobin and plasma nitrite concentration were closely correlated. Plasma chloride decreased initially with exposure to nitrite but quickly returned to control levels. Blue catfish exposed to nitrite at 10°C required 1 week to recover when placed in nitrite-free water. The methemoglobin reductase enzyme apparently functioned at a slow rate in fish acclimated to cold temperatures.     

Monoclonal Antibody-Based Analysis of the Renibacterium salmoninarum p57 Protein in Spawning Chinook and Coho Salmon

Abstract

Renibacterium salmoninarum, causative agent of bacterial kidney disease of salmonid fish, produces large amounts of soluble proteins during infection and broth culture. An enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies was developed for the precise quantification of p57, a major component of these proteins. Kidney, spleen, blood, and reproductive fluids of adult Pacific salmon Oncorhynchus spp. were examined by means of the assay. Kidney and spleen harbored the highest concentrations of this antigen. In the populations of returning salmon tested, 5–25% of the fish had p57 concentrations above a baseline level of 3 ng antigenig tissue, and antigen concentrations as high as 200 μg/g tissue were detected in kidneys of individual fish. The ELISA was compared to direct fluorescent antibody analysis, in which rabbit anti-R. salmoninarum antiserum was used to identify infected fish. There was 99% agreement (199 of 201 examined fish) between the two methods. Western blot analysis was used to demonstrate that antigenic proteins present in infected fish were similar to those seen from antigen prepared from broth culture of R. salmoninarum, although less degradation of p57 appears to occur in vivo.     

Comparative Uptake of Orally Administered Difloxacin in Atlantic Salmon in Freshwater and Seawater

Abstract

Fluoroquinolones are synthetic antibacterials with potential use for treatment of fish diseases. This study examined the comparative uptake of difloxacin, administered as a single oral gavage, by juvenile Atlantic salmon Salmo salar in freshwater and in seawater. In separate freshwater and seawater experiments with fish of the same spawning cohort, the concentration of difloxacin in plasma and fillets was determined at 1, 2, 4, 8, 12, 18, 24, and 48 h after the fish received the dose. Although plasma difloxacin concentration began to plateau in the freshwater fish at about 24 h, the highest average, 1,195 ng/mL, was in fish sampled at 48 h; thus, the plasma levels continued to rise through the end of the test. A similar pattern was observed in fillets from fish held in freshwater, but the peak average fillet concentration at 48 h was 4,737 ng/g. Contrastingly, in the seawater study, the average peak plasma concentration of 678 ng/mL was observed at 8 h after gavage and the peak average fillet concentration of 924 ng/g was observed at 18 h. Although difloxacin concentrations were higher in the freshwater-held fish than in the seawateradapted fish, the results suggest that difloxacin could be efficacious in treating bacterial diseases of Atlantic salmon in both freshwater and seawater.