Effect of lipopolysaccharides from Vibrio alginolyticus on the Mx gene expression and virus recovery from gilthead sea bream (Sparus aurata L.) experimentally infected with Nodavirus

Infections with nodavirus affect a wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to determine the immune status of gilthead sea bream that comes as a result of a Nodavirus infection, induced by activation of the interferon response pathway by lipopolysaccharides from Vibrio alginolyticus and the expression of interferoninduced Mx protein in liver samples. The enhancement of Mx protein gene expression was detected in liver samples of experimentally nodavirus infected fish and, furthermore, the immunostimulant LPS of V. alginolyticus decreased almost three times the virus titration with respect to no-immunized or infected with nodavirus group of fish.     

Sea bass Dicentrarchus labrax nervous necrosis virus isolates with distinct pathogenicity to sea bass larvae.

Reproduction of nodavirus disease was performed by experimental infection of sea bass eggs during fertilization or at larval stage 4 with 2 genetically distinguishable nodavirus strains (Sb1 and Sb2) isolated from sea bass collected along the Atlantic and Mediterranean French coast. The pathogenicity of the virus strains was assigned after detection of the virus by ELISA and immunohistochemistry (IHC). The Atlantic (Sb1) strain was more pathogenic than the Mediterranean (Sb2) strain during the fertilization step whilst both strains were pathogenic following experimental exposure of 4 d old larvae. Virus lesions developed in the brain 4 to 6 d following experimental exposure. Experimental ELISA proved very sensitive for detecting the nodavirus in Sb1 or Sb2 experimentally infected larvae, as well as in naturally infected sea bass larvae collected in French hatcheries or in barramundi larvae reared in the Pacific area. The development of an ELISA specific for the 2 nodavirus strains isolated from the sea bass should be useful for the detection of the virus, in addition to other techniques recommended by the Office International des Epizooties (OIE).     

Establishment,characterization and viral susceptibility of two cell lines derived from leopard wrasse Macropharyngodon geoffroy

Two new fish cell lines were established from skin (LWSK) and fin (LWFN) of leopard wrasse Macropharyngodon geoffroy. These cells grew optimally at 25° C in Leibovitz‐15 medium supplemented with 10% foetal bovine serum. Proliferation of M. geoffroy cells remained serum dependent up to cell passage 16, and cell‐plating efficiency ranged from 12 to 16%. Karyotypic analysis of these new cell lines at cell passage 8 indicated that both cell lines remained diploid with a peak chromosomal count of 144. PCR amplification of 16S mitochondrial DNA and the subsequent analysis confirmed that these cell lines were indeed derived from M. geoffroy. Results of viral challenge assays revealed that both LWSK and LWFN shared patterns of viral susceptibility similar to that of six fish viruses tested: LWSK and LWFN cells were highly permissive to channel catfish virus, spring viremia carp virus and snakehead rhabdovirus with high‐yield virus production ranging from 10^7·18±0·17 to 10^8·37±0·16 TCID₅₀ ml^?1 (mean ± s.d .). These newly established cell lines would be useful in attempts to isolate and study aquatic viruses, particularly the viral aetiology of green turtle fibropapilloma as M. geoffroy is known to be one of the common cleaner fish of green sea turtles.     

An automated microfluidic chip system for detection of piscine nodavirus and characterization of its potential carrier in grouper farms

Groupers of the Epinephelus spp. are an important aquaculture species of high economic value in the Asia Pacific region. They are susceptible to piscine nodavirus infection, which results in viral nervous necrosis disease. In this study, a rapid and sensitive automated microfluidic chip system was implemented for the detection of piscine nodavirus; this technology has the advantage of requiring small amounts of sample and has been developed and applied for managing grouper fish farms. Epidemiological investigations revealed an extremely high detection rate of piscine nodavirus (89% of fish samples) from 5 different locations in southern Taiwan. In addition, positive samples from the feces of fish-feeding birds indicated that the birds could be carrying the virus between fish farms. In the present study, we successfully introduced this advanced technology that combines engineering and biological approaches to aquaculture. In the future, we believe that this approach will improve fish farm management and aid in reducing the economic loss experienced by fish farmers due to widespread disease outbreaks.     

Sea bream Sparus aurata, an asymptomatic contagious fish host for nodavirus.

During an epidemiological survey of viral encephalopathy and retinopathy (VER) in diseased sea bass Dicentrarchus labrax, a nodavirus isolate was recovered from net pen-reared sea bream Sparus aurata harboured in the same farming premises. After the virus was isolated and identified by immunofluorescence on SSN-1 cells, sequence analysis with a PCR product from the T4 region of the capsid protein gene indicated that the virus shared 100% identity with a pathogenic virus strain isolated from sea bass. Infection trials demonstrated the pathogenicity of the sea bream virus isolate for juvenile sea bass whereas sea bream infected with the same virus isolate remained asymptomatic even following intramuscular injection of virus. Nevertheless, the sea bream appeared to be a potential carrier of nodavirus, as juvenile sea bass became infected when maintained in a tank containing experimentally contaminated sea bream.     

One‐step Multiplex RT‐PCR for Simultaneous Detection of Four Viruses in Tobacco

A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection.     

Detection of Puccinia pelargonii‐zonalis‐infected Geranium Tissues and Urediniospores

The occurrence of geranium rust (caused by Puccinia pelargonii‐zonalis) in commercial greenhouses can result in unmarketable plants and significant economic losses. Currently, detection of geranium rust relies solely on scouting for symptoms and signs of the disease. The purpose of this research was to develop a rapid detection assay for P. pelargonii‐zonalis‐infected tissues or urediniospores on greenhouse‐grown geraniums. Two oligonucleotide primers were designed based on internal transcribed spacer sequence data from three isolates of P. pelargonii‐zonalis. The primers amplified a 131‐bp product from genomic DNA from each isolate of P. pelargonii‐zonalis but did not amplify a product from genomic DNA from twelve other rust fungi or four other plant pathogenic fungi. A PCR product was amplified consistently from solutions that contained 1 ng or 100 pg/ml of purified P. pelargonii‐zonalis DNA in conventional PCR and at 1 pg/ml using real‐time PCR. The detection threshold was 10² urediniospores/ml for real‐time PCR and 10⁴ urediniospores/ml for conventional PCR using urediniospores collected by vacuum from sporulating lesions. Puccinia pelargonii‐zonalis DNA was amplified by real‐time PCR from urediniospores washed from a single inoculated leaf, but recovered urediniospores were below detection threshold from one inoculated leaf with 5, 10, 25 and 50 non‐inoculated leaves. Conventional and real‐time PCR did not detect P. pelargonii‐zonalis in infected leaf tissues, presumably due to PCR inhibitors in the geranium leaf tissue. The inhibition of both conventional and real‐time PCR by geranium tissues suggests that a detection assay focusing on urediniospore recovery and microscopic examination with subsequent species verification by PCR may be the most efficient method for assessing the presence of geranium rust in greenhouses.     

Quantifying Neodiprion sertifer nucleopolyhedrovirus DNA from insects,foliage and forest litter using the quantitative real‐time polymerase chain reaction

• 1 Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) is widely used as a viral bio‐insecticide against larvae of the European pine sawfly N. sertifer (Geoff.) (Hymenoptera: Diprionidae), which is one of the most harmful defoliators of pines in Northern Europe. A major obstacle to studying this pathogenic virus in nature is the difficulty of confirming and quantifying the presence of NeseNPV.
• 2 In the present study, we developed real‐time polymerase chain reaction (PCR) primers, based on the caspid gene 39 sequence, for the specific and quantitative detection of NeseNPV. The quantitative real‐time PCR (qPCR) assay can detect virus from any substrate tested, including different insect life stages (egg, larval, adult), pine foliage, and litter or ground vegetation. The reproducible detection limit for the real‐time assay is 0.013 pg of viral DNA (0.013×10^?12 g), corresponding to 136 viral genomes or approximately one to seven virus occlusion bodies per sample.
• 3 qPCR is a specific, quantitative, sensitive, reliable and flexible procedure, and is a good supplement to conventional microscopy‐ or bioassay‐based methods for detection of the virus. We have used qPCR to quantify the level of NeseNPV in samples collected in the field after aerial application of the virus, and demonstrated significantly higher virus levels in sawfly larvae from sprayed areas compared with unsprayed control areas 4 weeks after spraying.
• 4 This qPCR assay can be used to determine important aspects of the biology of NeseNPV (e.g. virus levels in different insect life stages and in their microhabitats on pine foliage and in forest litter).

     

A combined RT‐PCR and dot‐blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius)

Aims: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red‐spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. Methods and Results: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin‐labelled probes resulted in the identification of both genotypes separately. The application of the RT‐PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46·87% of viral nervous necrosis virus carriers. The combination of RT‐PCR and blot hybridization increases the detection rate up to 90·62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56·25%). Conclusions: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. Significance and Impact of the Study: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.     

Susceptibility of European sheatfish Silurus glanis to a panel of ranaviruses

The current study was undertaken in order to assess the risk that different ranaviruses might impose on European sheatfish aquaculture. As the European sheatfish virus (ESV) is a known pathogen causing losses in European sheatfish aquaculture, it was assumed that closely related exotic ranaviruses might also be able to infect European sheatfish and probably cause disease and mortality in this species. The differential susceptibility of European sheatfish (Silurus glanis) to various ranavirus isolates was assessed at two different temperatures (15°C and 25°C) in a recirculation system. Fish were infected experimentally with a panel of ranavirus isolates including ESV, European catfish virus (ECV), European catfish virus isolate 24 (ECV‐24), Epizootic haematopoietic necrosis virus (EHNV), Rana esculenta virus isolate Italy 282/ I02 (REV), short‐finned eel virus (SERV), Bohle iridovirus (BIV), guppy virus 6 (GV6), doctor fish virus (DFV) and Frog virus 3 (FV3). Significant mortalities were observed, as expected, in fish infected with ESV at 15°C (100%) as well as at 25°C (86/83%). Fish infected with ECV at 15°C showed no clinical signs of disease (8% mortality), whereas those fish infected at 25°C exhibited a cumulative mortality of 54%. Fatal disease was also induced by Italian isolate ECV‐24 at 25°C (81%). Virus isolates ESV, ECV and ECV‐24, generally the most genetically closely related viruses, were successfully isolated from dead fish by cell culture with subsequent identification by polymerase chain reaction (PCR) and sequence analysis. However, no mortality or clinical signs of disease were observed in the groups of sheatfish infected with the other ranaviruses investigated in the study, and none of those viruses were re‐isolated in cell culture or identified by PCR. It was concluded that European sheatfish are susceptible to infection with ESV, ECV and ECV‐24 under laboratory conditions, but not to infection with EHNV, REV, SERV, BIV, GV6, DFV or FV3. For ESV, the incubation period was shorter at 25°C compared to 15°C water temperature, but whereas all fish died after ESV infection at 15°C, some fish survived the infection at 25°C. Futhermore, the very young sheatfish were susceptible to ECV and ECV‐24 at 25°C, whereas there was no significant mortality in the group of older sheatfish challenged with ECV at 15°C. Therefore, the clinical characteristics of the disease seem to depend on the age of the fish as well as on the water temperature.     

Effects of the sea louse Lepeophtheirus salmonis on temporal changes in cortisol,sex steroids,growth and reproductive investment in Arctic charr Salvelinus alpinus

Groups of mature (5+ year old) Arctic charr Salvelinus alpinus held in sea water were exposed for 34 days to either a high (mean ±s.e . 0·15 ± 0·01 sea lice Lepeophtheirus salmonis g^?1 fish mass) (HI), medium (0·07 ± 0·00 sea lice g^?1 fish mass) (MI) or no [control (C)] sea‐lice infection during early stages of gonad development (June to July). Infection with sea lice resulted in increased plasma cortisol concentrations and this was related to intensity of infection; females tended to have higher cortisol concentrations than males at high infection intensities (HI group: female c. 130 ng ml^?1; male c. 80 ng ml^?1). Plasma osmolality (C c. 330, MI c. 350 and HI c. 415 mOsm) and chloride concentrations (C c. 135, MI c. 155 and HI c. 190 mM) increased significantly with infection intensity, indicating osmoregulatory problems in infected fish. A strong positive relationship between plasma osmolality and cortisol concentration was recorded. Plasma sex‐steroid concentrations were influenced negatively by sea‐lice infection, particularly in the HI group, and were inversely related to plasma cortisol concentrations. The most heavily infected fish postponed the initiation of reproductive development until exposed to fresh water and timing of ovulation tended to be delayed in these fish. Growth rate and condition were negatively influenced by sea‐lice infection and growth rate was inversely related to plasma cortisol concentrations. Sea‐lice infection resulted in mortality among females in the HI group, and the proportion of maturing females was lower in the MI group (46%) than in the controls (85%). Egg production in the MI and HI groups was c. 50 and 30% of the C group. Egg size, embryonic survival and fry mass did not differ across groups. Sea lice influence reproductive development and egg production in S. alpinus, and consequently these parasites may influence populations via sublethal effects on broodfish, affecting growth and condition, and their reproductive output.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.