Morphogenesis of the hatching gland of Atlantic halibut (Hippoglossus hippoglossus)

Summary The halibut hatching gland (HG) cells are first observed as a cellular disc in front of the embryonic head around the midpoint of intra ovo development. The disc is subsequently transformed into a loop of increasing diameter as the HG cells migrate over the anterior part of the yolk sac. When the HG disc is transformed into a loop, the density of HG cells is highest at the migratory front. Some HG cells lag behind the migrating front at the early stages of HG development. At maturity, all cells are contained in a narrow belt which is about 10 cells wide. The HG belt structure consists of a monolayer of HG cells, and is maintained while the cells migrate between the two epidermal cell layers. Migration is halted about 2 days before normal hatching when the HG cells reach a destination at about a right angle to on the embryonic axis. Under the scanning electron microscope, the differentiating HG cells protrude as a ridge the yolk sac surface. The HG cells immunostain with antiserum to hatching enzyme when the HG is observed as a crescent structure around the embryonic head. By counting the number of immunostaining cells in composite photos of the entire yolk sac membrane, we found that the HG belt consists of approximately 2000 secretory cells at maturity. This cell number stays fairly constant throughout the period of HG cell migration. Accordingly, mitoses of the halibut HG cells have generally ceased prior to morphogenesis, and cytodifferentiation is already quite advanced when cell migration starts. Offprint requests to: J.V. Helvik     

Viral encephalopathy and retinopathy (VER) in Atlantic salmon Salmo salar after intraperitoneal challenge with a nodavirus from Atlantic halibut Hippoglossus hippoglossus

Homogenate of tissue from juveniles of Atlantic halibut Hippoglossus hippoglossus suffering from viral encephalopathy and retinopathy (VER) was used to challenge smolt of Atlantic salmon Salmo salar with an initial average weight of 110 g. The nodavirus was administered in the form of an intraperitoneal injection, and the fish were kept for 134 d post challenge. Genotype characterisation of the nodavirus was performed by sequencing the RNA1 and RNA2 segments, and a quantitative real-time PCR (Q-PCR) assay was developed. Tissues from different organs were stained by immunohistochemistry (IHC). Samples were collected at random on Days 7, 25, 45, 69, 125 and 134 after challenge. Mortality, clinical signs and pathology of VER were observed only in the challenged group. The Q-PCR detected positive fish only in the challenged group, all of which were positive on all days of sampling. An increase in relative virus concentrations was observed from Day 7 to Day 25 post challenge. The increased level of virus concentration was maintained in the medulla oblongata throughout the experiment, suggesting persistence or slow elimination of the virus over time. The IHC detected positive cells on Days 34, 70 and 74. These results suggest that the nodavirus is transported to the medulla oblongata from the intraperitoneal injection site and is able to replicate in salmon. When injected, this nodavirus isolate caused mortality and established a persistent infection in the challenged salmon throughout the experiment. This susceptibility suggests that co-location of salmon and marine species should be avoided until further studies of possible transmission have been carried out.     

Age determination, bomb-radiocarbon validation and growth of Atlantic halibut (Hippoglossus hippoglossus) from the Northwest Atlantic

Atlantic halibut (Hippoglossus hippoglossus) is the largest and one of the most widely-ranging and commercially-valuable groundfish in the Atlantic Ocean. Although presumed to be long-lived, their age and growth has not been validated. Ages were estimated by counting growth increments from approximately 2400 thin-sectioned sagittal otoliths collected from the Scotian Shelf and southern Grand Banks off eastern Canada. The accuracy of age estimates made from otolith thin sections was validated using bomb-radiocarbon assays of 13 otolith cores whose year of formation ranged from 1949 to 1975, encompassing the timeframe of the global radiocarbon pulse. Known-age juvenile halibut from a culture facility were used to identify the approximate location of the first annulus. Growth rate for males and females was similar up to about 70 cm (~5 years), after which point male growth slowed, while female growth continued to an age of up to 38 years and a maximum observed size of 232 cm. Males grew to an observed maximum length of about 175 cm and a maximum age of 50 years. A comparison of age estimates for otoliths collected in a ‘historic’ time frame (1963 to 1974) with those from recent years (1997 to 2007) shows that growth rate has not changed appreciably between the two time periods. Small but significant growth differences were observed between the Scotian Shelf and southern Grand Banks for both sexes, while large differences in length at age were observed between halibut caught with longline compared to otter trawl due to differences in length-based gear selectivity. Age interpretations based on sectioned otoliths tended to be 10–15% different than those based on break and burn, although the age comparison was confounded by other variables and must be considered provisional. Atlantic halibut is a long-lived fish, living up to at least 50 years, an important consideration for the management of the fishery.     

Quantitative investigation of antigen and immune response in nervous and lymphoid tissues of Atlantic halibut (Hippoglossus hippoglossus) challenged with nodavirus

The present study reports the quantitative analysis of the spatio-temporal development of nodavirus infection and corresponding immune response in juvenile Atlantic halibut (Hippoglossus hippoglossus) challenged by intramuscular injection of nodavirus. Novel quantitative real-time RT-PCR protocols were applied to evaluate the absolute copy numbers of nodavirus RNA2 (RNA2) and secretory-IgM mRNA (sec-igmicro) in the eye, brain, mid/posterior kidney and spleen sampled over a period of 81 days. In the eye and brain, levels of both RNA2 and sec-igmicro increased significantly early in the infection. In the spleen and mid/posterior kidney, both RNA2 and sec-igmicro were detected but the levels remained unchanged during the experimental period. The levels of RNA2 and sec-igmicro in the eye and brain were strongly correlated (P<0.0001). Nodavirus antigen was demonstrated by immunohistochemistry (IHC) in the retina of eyes from a relatively few fish from day 34 post challenge (brain not examined), but not at any time in the spleen and anterior kidney. By IHC, IgM+ cells were observed in conjunction with nodavirus positive IHC labelling in the retina. In both the spleen and anterior kidney, the number of IgM+ cells increased from day 3 post challenge. By conventional real-time RT-PCR, RNA2 was only sporadically demonstrated in the posterior intestine, heart and gills. ELISA analysis revealed a nodavirus specific antibody response in serum that was significant from day 18 post challenge. No clinical signs or mortality related to nodavirus infection were observed in the challenged halibut. The results suggest that the nodavirus infection induced a significant antibody response through activation of B-cells in the kidney and spleen, and involved a substantial migration of antibody-secreting cells to infected peripheral tissues.     

Real-time PCR analysis of ovary- and brain-type aromatase gene expression during Atlantic halibut (Hippoglossus hippoglossus) development

Two forms of cytochrome P450 aromatase, acting in both the brain and the ovary, have been implicated in controlling ovarian development in fish. To better understand the expression of these two enzymes during sexual differentiation in Atlantic halibut (Hippoglossus hippoglossus), real-time PCR was used to quantify the mRNA levels of ovary- (cyp19a) and brain-type cytochrome P450 aromatase (cyp19b) genes in the gonad and brain during gonadal development. Both enzymes showed high levels of expression in both tissues in developmental stages prior to histologically detectable ovarian differentiation (38 mm fork length), with increased expression occurring slightly earlier in the brain than the gonad. Cyp19a showed a second peak of expression in later stages (> 48 mm) in the gonad, but not the brain. Cyp19b expression was generally higher in the brain than the gonad. These results suggest that sexual differentiation may begin in the brain prior to gonadal differentiation, supporting the idea that steroid hormone expression in the brain is a key determinant of phenotypic sex in fish. In an examination of sexually immature adults, cyp19a was highly expressed in female gonad while cyp19b was very highly expressed in the pituitary of both sexes. The ratio of cyp19a to cyp19b expression was much higher in ovaries than in testes in the adult fish, so this ratio was analyzed in the developing gonads of juvenile halibut in an attempt to infer their sex. This was only partially successful, with about half the fish in later developmental stages showing apparently sex-specific differences in aromatase expression.     

Serum immunoglobulin M in Atlantic halibut (Hippoglossus hippoglossus): characterisation of the molecule and its immunoreactivity

Three preparations of purified immunoglobulin (IgM) were isolated from serum of Atlantic halibut (Hippoglossus hippoglossus) by means of three different methods, and each of the three IgM preparations was used to produce a polyclonal rabbit anti-halibut IgM antiserum. One of the IgM preparations was employed in the characterisation of halibut serum immunoglobulin. Halibut IgM was shown to consist of two subunits, compatible with heavy (mu) and light (L) chains. A single mu chain at approximately 76 kDa, and six possible molecular weight (MW) variants of L chain were found (range approximately 25 to approximately 28.5 kDa). IgM was glycosylated on the heavy chain and N-linked carbohydrate constituted approximately 10.3% (w/w) of the total MW of IgM. The dominant form of non-reduced IgM had a MW of approximately 780 kDa, suggesting a tetrameric structure. Non-reduced IgM also showed a number of minor protein bands. Based on estimated MW, the relative carbohydrate content and the reactivity with all three anti-halibut IgM antisera, mono-, di- and trimeric redox forms of IgM were identified. The three antisera were characterised as to specificity and reactivity by means of enzyme linked immuno-sorbent assay (ELISA), crossed immuno-electrophoresis (CIE), and immunoblotting methods. The antisera showed a considerable diversity in their specificity to the suggested MW variants of halibut Ig light chain. A method for immunohistochemical detection of IgM in tissue was established. Protein A or protein G affinity for the IgM was not detectable.     

A study of the mechanism of algal uptake in yolk-sac larvae of Atlantic halibut (Hippoglossus hippoglossus)

The drinking rate of water, and the ingestion- and assimilation rates of algae in yolk-sac larvae of Atlantic halibut were examined by use of³H-dextran and¹⁴C-labelled algae. The drinking rate throughout the yolk-sac stage was in the range of 7–160 nl per larva h⁻¹, with a slight increase towards the end of the period. The ingestion rate of algae ( Tetraselmis sp.) was very low before day 30 and showed a peak between day 43 and 48 at 5° C. The assimilation efficiency of the algae was in the range of 1-5%. The clearance rate of algae was 100–1000 times higher than the drinking rate, and was correlated to the distance between the gill arches. Bigger cells were more effectively retained than smaller ones. The results suggested that the larvae acted as filter feeders from day 30 up to the point when they are expected to start feeding on bigger prey.     

Ten polymorphic microsatellite loci for the Atlantic halibut (Hippoglossus hippoglossus) and cross-species application in related species

In the present study, 10 polymorphic microsatellite DNA loci from Atlantic halibut (Hippoglossus hippoglossus) were isolated and characterized. The number of alleles for these loci ranged from 2 to 4 in tested 24 individuals. Observed and expected heterozygosities per locus varied from 0.21 to 0.70 and from 0.31 to 0.65, respectively. Most of these 10 microsatellite loci were successfully amplified and showed polymorphic in five related species. These loci will be useful for the assessment of genetic diversity and population structure of Atlantic halibut. H. Ding and C. Shao contributed equally to this work.     

Histomorphology of the early yolk-sac larvae of the Atlantic halibut (Hippoglossus hippoglossus L.)—an indication of the timing of functionality

Halibut larvae hatch at a very immature stage, and the duration of the yolk-sac period is very long (up to 50 days). This paper describes the histomorphological development of organs of the yolk-sac larvae (6° C) by use of light and electron microscopy. Rudimentary branchial cavities were open from 2 days after hatching. Kidneys seemed functional 16 days after hatching and onwards, and primitive lamellae on the gill arches were beginning to form at this age. Pancreatic zymogen granules were first observed 20daysafter hatching. The liver was segmented into lobes between 20 and 23 days after hatching, and the gall bladder seemed functional from day 23. The hindgut became extensively folded from day 26, and branchial capillaries were first observed at this stage. The larvae were able to catch food particles 24 days after hatching. Judging from ultrastructural observations, it seemed that halibut larvae were able to digest food particles between day 24 and 26 after hatching (around 150 daydegrees and 50% yolk absorption).     

Immune response to a recombinant capsid protein of striped jack nervous necrosis virus (SJNNV) in turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus, and evaluation of a vaccine against SJNNV.

Immunisation by intraperitoneal injection of an oil-emulgated recombinant partial capsid protein (rT2) from striped jack nervous necrosis virus (SJNNV) was performed on adult turbot Scophthalmus maximus and Atlantic halibut Hippoglossus hippoglossus. A specific humoral immune response was recorded in both species, and the levels of rT2-specific antibodies increased markedly in all groups during the 20 wk experiment. A challenge model for SJNNV was established by intramuscular injection of juvenile turbot. The turbot developed viral encephalopathy and retinopathy (VER), also known as viral nervous necrosis (VNN), with cumulative mortality in the range of 25 to 66%, after intramuscular inoculation with SJNNV propagated in the striped snake head cell line (SSN-1). Although neither clinical signs nor mortality were registered, SJNNV was neuroinvasive after bath exposure. The infection after both modes of challenge was verified by means of immunohistochemistry and RT-PCR, and SJNNV was reisolated in cell culture. The results indicate that SJNNV may have entered the central nervous system (CNS) by axonal transport through motor nerves after intramuscular inoculation. A vaccine efficacy test was performed on juvenile turbot, employing oil emulsified rT2 as a test vaccine and intramuscular inoculation of SJNNV. Significant protection was observed when the challenge was performed 10 wk post-vaccination.     

Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².     

Bath exposure of Atlantic halibut (Hippoglossus hippoglossus L.) yolk sac larvae to bacterial lipopolysaccharide (LPS): absorption and distribution of the LPS and effect on fish survival

Radiolabelled bacterial lipopolysaccharide (3H-LPS) obtained from Aeromonas salmonicida subsp. salmonicida was added to the petri dishes containing yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.). The larvae were exposed either to 6.25, 12.5, 25, 50 or 100 micrograms 3H-LPS ml-1. The uptake was both dependent on the LPS concentration and the time of exposure. After 5 days of exposure, each larva contained 1.8-7.4 ng 3H-LPS dependent on the initial concentration. After 10 days of exposure each larva contained 7.0-12.4 ng LPS and after 15 days they contained 18.3-34.9 ng 3H-LPS. Fluorescence microscopic analysis of sections obtained from larvae exposed to FITC-LPS (25, 50 and 100 micrograms ml-1) for 5, 10 and 15 days, revealed fluorescence in intestinal epithelial cells, cells in the connective tissue adjacent to the intestine, in cells located between the integumental layer and yolk sac, and in some epithelial cells in the integument. By use of immunohistochemical techniques, LPS was confined to intestinal epithelial cells, lumen of excretory duct and in numerous cells in the epidermal layer. Control specimens did not contain fluorescence or were immunohistochemically negative for LPS. In groups of larvae exposed to 12.5, 25, 50 and 100 micrograms LPS ml-1, the survival was significantly increased after exposure to 50 and 100 micrograms LPS ml-1 from day 20 (96 d degree) and throughout the yolk sac period compared to untreated larvae.     

A polymerase chain reaction (PCR) assay specific for Streptococcus suis based on the gene encoding the glutamate dehydrogenase

Polymerase chain reaction (PCR) primers that flank a 688-bp segment within the glutamate dehydrogenase gene (gdh) of Streptococcus suis type 2 could amplify efficiently the DNA of all 306 (100%) clinical S. suis isolates tested (pigs, n=305; human, n=1) encompassing all serotypes obtained from diverse organs, and geographic origins. When DNA from other bacteria were used as templates for amplification, no product was detected indicating specificity of the primers. Multiplex PCR was developed using the gdh gene primer pair and primers that targeted the gene encoding S. suis capsular biosynthesis (cps). This strategy enabled the detection of strains belonging to serotypes 1/2, 1, 2, 7, and 9, respectively. Using the multiplex-PCR technique, 12 out of 14 (86%) isolates that were previously identified as non-typable S. suis (based on biochemical reactions and serology) gave positive PCR results of which four were positive for serotype 7, three for serotype 2, and five for S. suis strains that belong to other serotypes. Retest results of all 14 isolates by several veterinary laboratories were identical with PCR and confirmed that the two non-PCR reactive isolates belonged to strains of other streptococcal species. These results indicated that PCR improved species determination and can thus be used as a reliable species-specific molecular diagnostic reagent for the accurate identification of S. suis isolates and a serotype-specific method for the detection of strains of serotypes 1/2, 1, 2, 7, and 9, respectively. The PCR method therefore has potential clinical and epidemiological applications.     

Comparison of the mouse antibody production (MAP) assay and polymerase chain reaction (PCR) assays for the detection of viral contaminants

Mouse antibody production (MAP) tests have become the standard assay for the detection of murine viral contamination in biologic materials, such as cell lines and transplantable tumors. However, newly developed PCR assays offer the advantage of lower cost, faster turn around times, and eliminate the use of live animals. In this study, the MAP test and a panel of PCR assays were compared for the detection of 11 different viral contaminants of cell lines and transplantable tumors. The PCR assays had either better or comparable results to the MAP test for all agents tested. The results of this study confirm that PCR assays are an effective method for detection of viral contamination and can be used as an alternative to the MAP test.     

Two-color allele-specific polymerase chain reaction (PCR-SSP) assay of the leptin receptor gene (Leprdb) for genotyping mouse diabetes mutation.

An allele specific polymerase chain reaction (PCR-SSP) assay for genotyping the mouse leptin receptor (Leprdb) mutation and its wild type (Lepr+) gene was developed using two different fluorescent dye-labeled primers. First, we determined the Leprdb and Lepr+ allele by PCR-SSP assay with usual dye-unlabeled primers. However this method requires two separate PCR reactions because the amplified products specific for each allele are almost the same size. We further developed a simple and reliable two-color PCR-SSP method that uses a color complementation strategy to distinguish the Leprdb and Lepr+ alleles. Leprdb/Leprdb, Leprdb/Lepr+ and Lepr+/Lepr+ of mice (5 each) were clearly genotyped by the two-color PCR-SSP. We also performed PCR-direct sequencing for the same samples and confirmed the accuracy of this method. This method makes it possible to reduce the number of PCR reactions because both alleles are amplified in the same reaction mixture.