Susceptibility of juvenile sole Solea senegalensis to marine isolates of viral haemorrhagic septicaemia virus from wild and farmed fish

The susceptibility of sole Solea senegalensis to infection with 3 viral haemorrhagic septicaemia virus (VHSV) strains obtained from wild Greenland halibut Reinhardtius hippoglossoides and farmed turbot Psetta maxima was demonstrated. Fish were infected by an intraperitoneal (i.p.), immersion or cohabitational route, and maintained at 16 degrees C. Infection trials showed that VHSV isolates were pathogenic for sole fingerlings by i.p. injection and waterborne exposure causing moderate levels of mortality (10 to 55%). In addition, the mortality observed in fish cohabitating with i.p.-infected sole confirms horizontal transmission of the virus. However, the low rates of mortality registered in this challenge suggest that there is a low dissemination of virus by the i.p.-infected sole, which results in lower secondary challenge of the cohabitating fish. External signs of disease included haemorrhaging of the ventral area and ascitic fluid in the body cavity. Dead fish were tested for VHSV by both cell culture and RT-PCR assay, using pools of kidney and spleen from 10 individuals. Virus was recovered from most of the pools composed of dead fish. The results obtained in this study not only demonstrate the susceptibility of sole to the VHSV strains employed but also indicate that wild VHSV marine isolates represent a potential risk for sole aquaculture.     

Susceptibility of juvenile Atlantic cod Gadus morhua to viral haemorrhagic septicaemia virus isolated from wild-caught Atlantic cod

A European strain of viral haemorrhagic septicaemia virus (VHSV) isolated from wild-caught cod Gadus morhua (H16/7/95) was shown to cause clinical disease and mortality in excess of 80% in juvenile Atlantic cod when administered by the intra-peritoneal (i.p.) route. No virus was recovered from cod cohabiting with experimentally infected fish at a ratio of 1:1, and no VHSV-associated mortality was demonstrated following immersion infection. External signs of disease in cod were the presence of exophthalmia and ascites. Virus was identified as VHSV by enzyme-linked immunosorbent assay (ELISA) and was recovered from both brain and organ pools (kidney, liver and spleen) of 100% of i.p. infected cod mortalities. Virus was also detected using an indirect immunofluorescence test on tissue imprints of kidney, liver, spleen and brain taken from moribund fish. The fact that cod were not susceptible to VHSV following waterborne exposure raises important questions surrounding the propagation, maintenance and impact of a naturally occurring reservoir of virus in the marine environment.     

Experimental transmission of Enteromyxum scophthalmi (Myxozoa), an enteric parasite of turbot Scophthalmus maximus

Several experiments were designed to elucidate the modes of transmission of the myxozoan parasite Enteromyxum scophthalmi to turbot Scophthalmus maximus. Direct transmission of the infections was achieved by cohabitation of infected and test fish, through waterborne contamination from the effluent of a tank containing infected fish, and via the oral route using parasite-infected intestines. The transmission of the turbot enteromyxosis was successful in all the fish exposed to the parasite by the 3 routes; accumulated mortality reached 100% at the end of most experiments. The progress of the infections was monitored by study of the histopathology. Influence of the mode of exposure was observed, with the oral route the fastest to initiate the parasite infections. The temperature also affected the course of the infections, which were established earlier at higher water temperature. Direct fish-to-fish transmission of the disease explains the rapid spreading of the turbot enteromyxosis in farms.     

Genotyping and pathogenicity of viral hemorrhagic septicemia virus from free-living turbot (Psetta maxima) in a Turkish coastal area of the Black Sea

Viral hemorrhagic septicemia (VHS) is one of the most serious fish viral diseases for cultured rainbow trout (Oncorhynchus mykiss), although VHS virus (VHSV) seems to be ubiquitous among marine fishes. In the present study, VHSV isolation was performed with free-living and cultured turbot (Psetta maxima) in the Trabzon coastal area of the Black Sea to evaluate participation of VHSV in mass mortalities of seed-produced turbot larvae. VHSV was detected in 14 of 66 free-living spawners (positive ratio, 21.2%), 1 of 65 free-living immature fish (1.5%) and 7 of 40 cultured brood stock (17.5%), respectively. Based on a partial glycoprotein gene nucleotide sequence, Turkish VHSV isolates were classified into the class I-e of genotype I and were the most closely related to the GE-1.2 isolate (>98% identity), which was found >20 years ago in Georgia. Thus, it was revealed that Turkish VHSV isolates were not introduced from European countries, it could be an indigenous type of VHSV distributing in the Black Sea environment. In pathogenicity tests, the Turkish isolates did not induce mortality in turbot larvae and rainbow trout fingerlings. Mass mortalities at a rate of approximately 90% occurred in turbot larvae produced by experimental seeding, although VHSV was not detected in any dead fish. Thus, it was concluded that mass mortality in the seed-produced turbot larvae was not caused by VHSV infection.     

Mycobacterial infection in farmed turbot Scophthalmus maximus

Mycobacteriosis (piscine tuberculosis) has been reported to affect a wide range of freshwater and marine fish species; however, this is the first report describing mycobacterial infections in turbot Scophthalmus maximus. High numbers of granulomas were initially observed in the organs of moribund farmed turbot. Bacteriological analysis of organs with granulomas led to the isolation of Mycobacterium marinum. Further analysis, to determine the prevalence of the infection in the farm and to identify its source, showed the occurrence of a dual infection by M. marinum and M. chelonae. The presence of Nocardia sp. in some of the fish infected with mycobacteria was also detected. The presence of granulomas in internal organs of apparently healthy fish indicated a high prevalence of the disease, a conclusion that was supported by isolating mycobacteria from all fish with or without granulomas. The infection was probably responsible for the mortality observed (approximately 2% mo(-1)), as most of the recently dead fish presented high numbers of granulomas and isolation of mycobacteria was possible from all of the fish. The isolation of M. marinum from the inlet water suggested this as the most plausible source for the infection occurring in the farm.     

Comparative susceptibility of turbot Scophthalmus maximus to different genotypes of viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) disease has exerted a significant impact on the development of turbot aquaculture in the British Isles. The source of such outbreaks is believed to be naturally occurring marine isolates of viral haemorrhagic septicaemia virus (VHSV), which are endemic in the marine environment of Northern Europe. Genetic studies have classified these marine VHSV isolates into genotypes based on their geographic rather than host-species origin. This study set out to explore the hypothesis that susceptibility of turbot to VHSV might be genotype specific. Immersion infection of turbot with a range of isolates, selected according to genotype, identified significant differences between susceptibility and genotype. Viruses belonging to Genotypes Ib (Baltic marine isolates) and III (North Sea/E. Atlantic marine isolates) caused significantly higher mortality than isolates from Genotypes Ia (isolates associated with rainbow trout aquaculture) and II (Baltic marine isolates). This study serves to highlight the importance of thoroughly investigating the susceptibility of any given species to the range of pathogens to which they might be exposed prior to considering them resistant to any disease. Furthermore, it highlights different risk factors that might be associated with turbot aquaculture undertaken in different environments. Finally, an increased knowledge of the relative virulence of different isolates in turbot will assist in understanding virulence determinants, which could lead to advances in disease control.     

蟹栖异阿脑虫寄生大菱鲆及其组织病理学研究

论文首次报道了蟹栖异阿脑虫(Mesanophrys carcini)在养殖大菱鲆体内的寄生情况以及所引起宿主的组织病理学变化。观察结果表明,感染的苗期大菱鲆的大部分器官组织包括皮肤、鳍、鳃、眼、脑、心脏、肝脏、肾脏、脾脏、胰腺和消化道等以及器官组织间隙中都发现了寄生的蟹栖异阿脑虫,属于全身性感染;而对于成鱼,蟹栖异阿脑虫主要寄生于皮肤、鳍和鳃等器官组织;严重感染者,蟹栖异阿脑虫可侵入其脑、肝脏、肾脏和心脏等器官组织,但数量不多。蟹栖异阿脑虫在大菱鲆体内的寄生能够引起病鱼各器官组织发生不同程度的组织病理学改变。该病在幼鱼和成鱼身上所表现出较一致的病理学特征为:鳃小叶呼吸上皮细胞增生;脑膜炎和脑组织液化性坏死;肾脏和脾脏造血组织坏死;肝脏中央静脉和窦状隙扩张;肌纤维发生凝固性坏死和眼的脉络膜充血、水肿;单核巨噬细胞浸润。研究认为,蟹栖异阿脑虫寄生引起的鳃损害使病鱼呼吸困难,窒息是病鱼发生死亡的主要原因,而虫体寄生导致的各器官组织变性,坏死,使得病鱼器官功能衰竭可加速鱼的死亡。另外,文中还探讨了蟹栖异阿脑虫入侵大菱鲆的可能途径,认为破损的皮肤和鳃是入侵的主要途径。研究结果告诉我们,在大菱鲆养殖过程中防止鱼体擦伤,保持池底清洁对于预防疾病的发生非常重要;对该病的治疗应采取外部杀虫和内部口服药物相结合的方式。     

Host range susceptibility of Enterococcus sp. strains isolated from diseased turbot: possible routes of infection.

Experiments were conducted to assess the pathogenicity of Enterococcus sp. strains isolated from diseased turbot for several fish species (turbot, salmon, trout, and seabream), as well as for mice. The intraperitoneal injection assays indicated that the tested strains showed host specificity for turbot, with a high degree of virulence (50% lethal dose of 10(4) cells per g of fish). The Spanish Enterococcus sp. isolates were nonpathogenic for the other fish species studied and for mice. The possible routes of infection were determined by bath exposure (with and without prior abrasion of the skin) and by intragastric inoculations with food and feces contaminated with the pathogen. The bath challenges indicated that the Enterococcus isolates were able to overcome the defense mechanisms present on the surface of the turbot only if the skin was abraded prior to the exposure. The antibacterial activities of components of a glycoprotein nature present in the turbot skin mucus are probably responsible in part for the resistance in noninjured fish to infection. On the other hand, we demonstrated the capacity of this pathogen to overcome adverse conditions in the stomachs of fish when associated with food or fecal material, since it is able to establish an infective state and to produce mortalities after 16 to 20 days postingestion. From all of these findings, we can conclude that horizontal transmissions through water and the fecal-oral route are the main avenues of infection of turbot streptococcosis.     

Physiological responses to handling in the turbot

Turbot Scophthalmus maximus were cannulated via the afferent branchial artery and were either net-confined in sea water or in air for 9 min to monitor the stress response of a hatchery-reared marine flatfish. No mortality was observed. Aerial exposure appeared to mobilize plasma free fatty acids and stimulate the interrenal tissue but had no effect on circulating glucose or lactate levels. This pattern was qualitatively similar to that induced by net-confinement in sea water, although the magnitude and duration of the changes were more marked in the turbot handled in sea water. Aerial exposure had no effect on plasma osmolality, protein, or Cl⁻ concentrations and only a minor effect on plasma Na⁺ concentrations, which was in sharp contrast to the ionoregulatory disturbance noted in turbot which were net-confined in sea water. However, plasma K⁺ and cortisol concentrations were similarly elevated by both handling procedures. The results from these experiments suggest that whilst similarities with the salmonid physiological stress response are apparent, the lack of a plasma glucose response may represent a fundamental difference in turbot stress physiology.     

Microarray-Based Identification of Differentially Expressed Genes in Families of Turbot (Scophthalmus maximus) After Infection with Viral Haemorrhagic Septicaemia Virus (VHSV)

Viral haemorrhagic septicaemia virus (VHSV) is one of the major threats to the development of the aquaculture industry worldwide. The present study was aimed to identify genes differentially expressed in several turbot (Scophthalmus maximus) families showing different mortality rates after VHSV. The expression analysis was conducted through genome-wide expression profiling with an oligo-microarray in the head kidney. A significant proportion of the variation in the gene expression profiles seemed to be explained by the genetic background, indicating that the mechanisms by which particular species and/or populations can resist a pathogen(s) are complex and multifactorial. Before the experimental infections, fish from resistant families (low mortality rates after VHSV infection) showed high expression of different antimicrobial peptides, suggesting that their pre-immune state may be stronger than fish of susceptible families (high mortality rates after VHSV infection). After infection, fish from both high- and low-mortality families showed an up-modulation of the interferon-induced Mx2 gene, the IL-8 gene and the VHSV-induced protein 5 gene compared with control groups. Low levels of several molecules secreted in the mucus were observed in high-mortality families, but different genes involved in viral entrance into target cells were down-regulated in low-mortality families. Moreover, these families also showed a strong down-modulation of marker genes related to VHSV target organs, including biochemical markers of renal dysfunction and myocardial injury. In general, the expression of different genes involved in the metabolism of sugars, lipids and proteins were decreased in both low- and high-mortality families after infection. The present study serves as an initial screen for genes of interest and provides an extensive overview of the genetic basis underlying the differences between families that are resistant or susceptible to VHSV infection.     

Quantitative and qualitative evaluation of iNOS expression in turbot (Psetta maxima) infected with Enteromyxum scophthalmi

Enteromyxum scophthalmi is the causative agent of turbot enteromyxosis, an intestinal parasitisation that produces severe desquamative enteritis leading to a cachectic syndrome and eventually the death. It is well known the importance of the innate immune response against parasites in fish, with the release of antimicrobial substances such as reactive oxygen and nitrogen species, produced by the inducible nitric oxide synthase (iNOS). This enzyme is mainly found in phagocytes, but also in structural cells from the intestinal mucosa. The aim of this study was to characterize iNOS in intestine and lymphohaematopoietic organs (spleen and anterior kidney) of turbot by means of immunohistochemistry in order to assess the possible changes of this enzyme through the infection. The presence of the enzyme was evaluated in control and E. scophthalmi-infected turbot. The results showed immunoreactivity in the apical border of enterocytes and mild staining of goblet cells in both control and infected turbot although it was more evident and widespread in infected turbot compared to control. Moderate numbers of iNOS+ cells were present in the lamina propria-submucosa of fish which presented moderate and severe inflammatory infiltrates at this level. In spleen and kidney, iNOS+ cells were scattered through the parenchyma and, in severely infected fish, tended to be allocated near the vascular structures and melano-macrophage centres. The number of positive cells at the lymphohaematopoietic organs was significantly higher in infected turbot and increased as infection progressed. The increase in the expression of iNOS in the tissues of E. scophthalmi-infected turbot was more evident in individuals with severer lesions. The measurement of the levels of iNOS during turbot enteromyxosis reveals a possibly delayed response that would not able to eliminate the parasites but would exacerbate mucosal injury.     

Viral hemorrhagic septicemia virus alters turbot Scophthalmus maximus macrophage nitric oxide production.

The effect of viral hemorrhagic septicemia virus (VHSV) in vitro infection on the nitric oxide (NO) production by turbot Scophthalmus maximus kidney macrophages has been addressed in the past. Previously, we had determined that only a small fraction of turbot possess head kidney macrophages that respond to a single exposure of lipopolysaccharide (LPS) with NO production (LPS-responsive macrophages), whereas macrophage cultures from other individuals were not activated by LPS alone and needed a combination of stimuli to respond (LPS-non-responsive macrophages). In the current work, we examined the effect of VHSV on NO production by macrophages characterized as LPS-responsive macrophages or LPS-non-responsive macrophages. Combinations of LPS and tumor necrosis factor alpha (TNF-alpha) and macrophage-activating factor (MAF) were also used to stimulate the cells for NO production. The effect of VHSV on NO production depends on the response to LPS alone. When a low multiplicity of infection was used (1.78 x 10(-3)), the NO production in response to LPS in LPS-responsive macrophages was significantly decreased. However, LPS-non-responsive macrophage cultures produced NO when a combination of LPS and VHSV was used. In the case of a higher VHSV multiplicity of infection (1.78), no significant change was observed in LPS-non-responsive animals. Combinations of LPS with TNF-alpha, LPS with MAF, and TNF-alpha with MAF were used to induce NO production in LPS-non-responsive macrophages. In all these cases, VHSV suppressed NO production, although at a significant level only when a combination of TNF-alpha and MAF was used for the induction of NO.     

Development of an effective Edwardsiella tarda vaccine for cultured turbot (Scophthalmus maximus)

Since 2004 Edwardsiella tarda has become one of the most important emerging pathogens in turbot aquaculture industry in Europe causing serious economic losses. Therefore, this study aimed to design an effective vaccination strategy to prevent edwardsiellosis in this fish species. Two vaccine formulations, an adjuvanted vaccine and an aqueous bacterin, and different routes of administration, bath and intraperitoneal injection (i.p.), were tested. The effectiveness of the different immunization strategies was evaluated in terms of relative percent survival (RPS) and antibody levels. On the basis of the results obtained we recommend the i.p. administration of a non-mineral oil adjuvanted vaccine via i.p., which confers RPS values over 90% at least 6months post-vaccination.     

A Reverse Genetics System for the Great Lakes Strain of Viral Hemorrhagic Septicemia Virus: the NV Gene is Required for Pathogenicity

Viral hemorrhagic septicemia virus (VHSV), belonging to the genus Novirhabdovirus in the family of Rhabdoviridae, causes a highly contagious disease of fresh and saltwater fish worldwide. Recently, a novel genotype of VHSV, designated IVb, has invaded the Great Lakes in North America, causing large-scale epidemics in wild fish. An efficient reverse genetics system was developed to generate a recombinant VHSV of genotype IVb from cloned cDNA. The recombinant VHSV (rVHSV) was comparable to the parental wild-type strain both in vitro and in vivo, causing high mortality in yellow perch (Perca flavescens). A modified recombinant VHSV was generated in which the NV gene was substituted with an enhanced green fluorescent protein gene (rVHSV-ΔNV-EGFP), and another recombinant was made by inserting the EGFP gene into the full-length viral clone between the P and M genes (rVHSV-EGFP). The in vitro replication kinetics of rVHSV-EGFP was similar to rVHSV; however, the rVHSV-ΔNV-EGFP grew 2 logs lower. In yellow perch challenges, wtVHSV and rVHSV induced 82-100% cumulative per cent mortality (CPM), respectively, whereas rVHSV-EGFP produced 62% CPM and rVHSV-ΔNV-EGFP caused only 15% CPM. No reversion of mutation was detected in the recovered viruses and the recombinant viruses stably maintained the foreign gene after several passages. These results indicate that the NV gene of VHSV is not essential for viral replication in vitro and in vivo, but it plays an important role in viral replication efficiency and pathogenicity. This system will facilitate studies of VHSV replication, virulence, and production of viral vectored vaccines.     

Virulence and nucleotide sequence analysis of marine viral haemorrhagic septicaemia virus following in vivo passage in rainbow trout Onchorhynchus mykiss

A marine isolate of viral haemorrhagic septicaemia virus (VHSV) (860/94) was passaged in triplicate through sequential batches of rainbow trout via an intra-peritoneal infection route, without amplification in tissue culture. Following 5 passages, the VHSV glycoprotein gene was amplified directly from fish tissue homogenates and the consensus sequence compared to that of the original tissue culture isolate. Virus was also recovered directly from pools of kidney and spleen material after 5 passage events, and its virulence compared to that of unpassaged material by intra-peritoneal infection. Following passage in rainbow trout, isolate 860/94 exhibited a higher virulence for rainbow trout than unpassaged material. Sequence comparisons identified no difference in the consensus sequence of the glycoprotein gene following in vivo passage. The mechanisms responsible for the observed increase in virulence of isolate 860/94 following passage in rainbow trout thus remain unknown. The possibility that viral isolates may exhibit an increased virulence following passage in novel host species does, however, have important implications with regard to the epidemiology of this important fish pathogen.     

Drinking and water absorption by the larvae of herring (Clupea harengus) and turbot (Scophthalmus maximus)

The efficiencies of water absorption from the guts of the larvae of herring ( Clupea harengus L.) and turbot ( Scophthalmus maximus L.) were estimated by two methods, The first method was based on the differences in the rates of accumulation, by drinking, and clearance from the gut of radiolabelled inert markers. The second method used the equilibrium level of radioactivity in the larvae to measure the concentration of the markers in the gut above background as water is absorbed from the gut. Water absorption efficiencies for herring larvae drinking sea water were estimated to be 77% using both methods. When external salinity was reduced to 50% sea water, drinking rates and water absorption efficiency in herring larvae fell substantially.
Estimates of water absorption efficiency of seawater-adapted turbot larvae were similar (71–84%) to that of herring using both methods. Although temperature had a marked effect on both the rate of drinking and water absorption, there was no significant thermal effect on the efficiency of absorption from the guts of turbot larvae. The limitations of the techniques and the implications of the estimates in terms of water balance in fish larvae are discussed.     

A comparative study of blood oxygen transport in turbot and sea bass: effect of chronic hypoxia

A comparative study of blood oxygen binding and carrying capacities of turbot Scophthalmus maximus and sea bass Dicentrarchus labrax , two fish species differing in their demand for oxygen, was carried out under three levels of chronic hypoxia ( P o ₂ = 93, 65 and 40 mmHg) for 40 days. Blood O₂ affinity in normoxia was moderately high in both species ( P ₅₀ was c . 12–13 mmHg at pH 7·7). The Bohr factor was significantly lower in turbot (−0·52) than in sea bass (−0·85). In both species, blood O₂ affinity was not significantly affected by oxygen depletion whatever its level and duration. In turbot, however, P ₅₀ appeared to slightly decrease at the two more severe levels of hypoxia. In both species, blood O₂ carrying capacity was not affected by hypoxia and remained twice as high in sea bass than in turbot.     

Novel horizontal transmission route for Enteromyxum leei (Myxozoa) by anal intubation of gilthead sea bream Sparus aurata

The aim of the present study was to determine whether Enteromyxum leei, one of the most threatening parasitic diseases in Mediterranean fish culture, could be transmitted by peranal intubation in gilthead sea bream Sparus aurata L. Fish were inoculated either orally or anally with intestinal scrapings of infected fish in 3 trials. Oral transmission failed, but the parasite was efficiently and quickly transmitted peranally. Prevalence of infection was 100% at 60 d post inoculation (p.i.) in Trial 1 under high summer temperature (22 to 25 degrees C; fish weight = 187.1 g), and 85.7% in just 15 d p.i. in Trial 3 using smaller fish (127.5 g) at autumn temperature (19 to 22 degrees C). In Trial 2, prevalence reached 60% at 60 d p.i. in the group reared at constant temperature (18 degrees C), whereas no fish was infected in the group that was kept at low winter temperature (11 to 12 degrees C), although infection appeared (46.1% at 216 d p.i.) when temperature increased in spring. The arrested development at low temperature has important epidemiological consequences, as fish giving false negative results in winter can act as reservoirs of the parasite. Histopathological examination showed a posterior-anterior intestinal gradient in the progression of the infection, in terms of both intensity and parasite maturation. Thus, per-anal intubation provides a very uniform, reliable and faster mode of transmission of E. leei than the commonly used transmission methods (cohabitation, exposure to infected effluent and oral inoculation), which require long exposure times or give variable and unpredictable results.