In vitro infection of a cell line from Ictalurus nebulosus with Piscirickettsia salmonis.

Piscirickettsia salmonis, the etiologic agent of salmonid rickettsial septicemia (SRS), affects several species of salmonids. Previous reports using the appearance of cytopathic effect (CPE) as the criterion for susceptibility, showed that Piscirickettsia salmonis (ATCC strain) can be grown in vitro in some cells lines derived from salmonid fish, but not in BB cells from brown bullhead (Ictalurus nebulosus) and BF-2 cells from bluegill (Lepomis macrochirus). In this study we describe growth of P. salmonis (ATCC strain VR 1361) in a cell line previously believed to be nonpermissive for this organism. CPE was first detected in chinook salmon embryo (CHSE-214) and epithelioma papulosum ciprini (EPC) cell lines at 6 d postinfection (dpi). In contrast, using BB cell line, CPE was first detected 45 dpi and the monolayer completed CPE by 78 dpi. Electron microscopic examination of BB cells 78 dpi revealed free, intracytoplasmic and extracellular localization of the agent. P. salmonis was also observed within membrane-bounded vacuoles in BB cells, similar to that described in CHSE 214 cells. Contrary to earlier reports, results from the present study show that the BB cell line, is susceptible to Piscirickettsia salmonis infection. The delayed onset of CPE in BB cells in comparison to other permissive cell lines suggests that BB cells are not ideal hosts for P. salmonis. Interestingly, however, these results demonstrate that P. salmonis can infect non-salmonid cell lines, and raises the possibility that non-salmonid fish may play a role in the persistence and transmission of SRS in the natural environment.     

Culture of Piscirickettsia salmonis on enriched blood agar.

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, an economically significant disease of fish. Isolation of P. salmonis by culturing on fish cell lines has been the standard technique since the initial isolation of the organism. The ability to grow P. salmonis on artificial media would relieve facilities of the cost of maintaining cell lines, permit isolation at fish culture sites with fewer contamination problems, and allow easier transport of isolates to diagnostic facilities for confirmation assays. This report describes the successful culture of P. salmonis on enriched blood agar.     

Amplification of a Piscirickettsia salmonis-like 16S rDNA Product from Bacterioplankton DNA Collected from the Coastal Waters of Oregon,USA

Abstract

Piscirickettsiosis is a disease observed in salmonids reared in net-pens in the North and South Pacific and in the North Atlantic oceans. Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a gram-negative, obligate, intracellular pathogen that replicates within cytoplasmic vacuoles. We describe here the detection, by dot-blot hybridization, polymerase chain reaction, and sequence analysis, of a 16S rDNA sequence that is 99.5–98.2% similar to the 16S rDNA sequences of known isolates of P. salmonis.     

Comparative evaluation of molecular diagnostic tests for Nucleospora salmonis and prevalence in migrating juvenile salmonids from the Snake River, USA

Nucleospora salmonis is an intranuclear microsporidian that primarily infects lymphoblast cells and contributes to chronic lymphoblastosis and a leukemia-like condition in a range of salmonid species. The primary goal of this study was to evaluate the prevalence of N. salmonis in out-migrating juvenile hatchery and wild Chinook salmon Oncorhynchus tshawytscha and steelhead O. mykiss from the Snake River in the U.S. Pacific Northwest. To achieve this goal, we first addressed the following concerns about current molecular diagnostic tests for N. salmonis: (1) nonspecific amplification patterns by the published nested polymerase chain reaction (nPCR) test, (2) incomplete validation of the published quantitative PCR (qPCR) test, and (3) whether N. salmonis can be detected reliably from nonlethal samples. Here, we present an optimized nPCR protocol that eliminates nonspecific amplification. During validation of the published qPCR test, our laboratory developed a second qPCR test that targeted a different gene sequence and used different probe chemistry for comparison purposes. We simultaneously evaluated the two different qPCR tests for N. salmonis and foundthat both assays were highly specific, sensitive, and repeatable. The nPCR and qPCR tests had good overall concordance when DNA samples derived from both apparently healthy and clinically diseased hatchery rainbow trout were tested. Finally, we demonstrated that gill snips were a suitable tissue for nonlethal detection of N. salmonis DNA in juvenile salmonids. Monitoring of juvenile salmonid fish in the Snake River over a 3-year period revealed low prevalence of N. salmonis in hatchery and wild Chinook salmon and wild steelhead but significantly higher prevalence in hatchery-derived steelhead. Routine monitoring of N. salmonis is not performed for all hatchery steelhead populations. At present, the possible contribution of this pathogen to delayed mortality of steelhead has not been determined.     

Influence of reduced feed ration on Lepeophtheirus salmonis infestation and inflammatory gene expression in juvenile pink salmon

The effect of reduced feed ration on infestation levels with the sea louse Lepeophtheirus salmonis and gene expression in juvenile pink salmon Oncorhynchus gorbuscha was tested in three laboratory trials. Body weight was significantly lower among fish on the reduced ration for 27, 34, or 65 d than fish on the full ration. Neither the prevalence nor the abundance of L. salmonis differed between fish on full and reduced rations at any time in any trial. In trial 2, sea louse rejection was delayed among fish on reduced rations; however, the parasite was ultimately rejected from all fish in this trial regardless of ration. Proinflammatory gene expression in salmon exposed to L. salmonis was modulated by reduced rations. There was a reduction in the expression of interleukin-8 in pink salmon on reduced rations 7 d after exposure but not 14 d after exposure. In contrast, the 7-d expression of interleukin-1 beta (IL-1beta) was reduced in exposed pink salmon regardless of ration. By day 14, however, expression of IL-1beta was increased in association with reduced rations among exposed salmon. Similarly, the expression of tumor necrosis factor alpha (TNFalpha) was increased 14 d after exposure among salmon on a reduced ration. There was no evidence that short-duration exposure of otherwise healthy juvenile pink salmon to a reduced ration affected susceptibility to L. salmonis. The expression data do not suggest an obvious mechanism of louse rejection; rather, they indicate that a more comprehensive suite of inflammatory pathways should be surveyed to better understand the early pink salmon response to L. salmonis.     

The Gill Pathogen Dermocystidium salmonis in Oregon Salmonids

Abstract

Intense infections of the gill pathogen Dermocystidium salmonis were associated with mortality of prespawning chinook salmon Oncorhynchus tshawytscha in several Oregon rivers in 1988. The occurrence of the pathogen in returning adult chinook salmon was monitored in several coastal Oregon stocks from 1989 to 1993. Although the prevalence of the pathogen was high in these fish (up to 66.6%), infection intensities were generally low, and no mortality attributable to D. salmonis was observed. In 1988, the pathogen was associated with a lethal epizootic among juvenile chinook salmon smolts at the Trask State Fish Hatchery near Tillamook, Oregon. Histological examination of gills from heavily infected fish revealed hyperplasia of gill epithelium and fusion of gill lamellae. When naturally infected smolts were transferred from fresh to salt water, the most heavily infected fish died within 10 d, and the number of D. salmonis cysts declined and disappeared from previously infected salmon after 21–42 d.     

Piscirickettsiosis and piscirickettsiosis-like infections in fish: a review

Piscirickettsia salmonis was the first "rickettsia-like" bacteria to be recognized as a pathogenic agent of fish. Since the first reports of piscirickettsiosis emerged from Chile in the late 1980s, Piscirickettsia-like bacteria have been recognized with increasing frequency in a variety of fish species, from both fresh and saltwaters around the world. Although the first reported incidents of Piscirickettsia were in salmonids, Piscirickettsia-like bacteria are now being frequently associated with disease syndromes in non-salmonid fish. Mortalities have occurred in white seabass (Atactoscion noblis), black seabass (Dicentrarchus sp.), tilapia (Oreochromis, Tilapia and Sarotherodon spp.) and blue-eyed plecostomus (Panaque suttoni). Piscirickettsiosis and piscirickettsiosis-like diseases have affected aquaculture productivity, profitability, the species of fish compatible with commercial rearing, and transportation of fish from site to site. Piscirickettsiosis and syndromes caused by similar bacteria are an emerging disease complex that will increasingly inhibit fish production.     

Sequential pathology of the gills of Coho salmon with a combined diatom and microsporidian gill infection

A large group of 40 gram Coho salmon smolts experienced 60% mortality within the first week after introduction to cages in sea-water. Histological and ultrastructural examination of sequential samples of gill tissue revealed a dramatic suppurative branchitis accompanied by extensive fusion of gill lamellae. This dramatic host response and subsequent high mortality appeared to be in response to a bloom of Corethron-like diatoms. A further complication was an extensive infection of endothelial cells of the gill vasculature by a microsporidian protozoan.     

MHC class II+ cells in the gills of Atlantic salmon (Salmo salar L.) affected by amoebic gill disease

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II β chain. A tank housing Atlantic salmon (Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II β chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II⁺ cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II⁺ cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II⁺ cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II⁺ cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.     

Early interactions of Edwardsiella ictaluri, with Pangasianodon catfish and its invasive ability in cell lines

Commercial Pangasianodon catfish production is heavily impacted by Bacillary Necrosis of Pangasius (BNP) caused by Edwardsiella ictaluri. This study aimed to investigate the early bacterium-host interactions following immersion challenge and to compare the retrieved data with the invasion ability of the used isolates in fish cell lines. Firstly, Pangasianodon hypophthalmus fingerlings were challenged via immersion using E. ictaluri isolate HO2 or 223. At different times post inoculation, fish were sacrificed and gill and internal organ samples were taken for bacteriological, histological and immunohistochemical evaluation. The bacterial load was higher for fish inoculated with isolate HO2 compared with 223. Histological and immunohistochemical analysis revealed multifocal necrotic areas in kidney, spleen and liver of HO2 inoculated fish at 72 h post inoculation with short rod-shaped immunoperoxidase positive bacteria clustered inside cells respectively. Bacteria especially were present in the gills and intestinal tract of HO2 inoculated fish, suggesting the gastrointestinal tract and gills act as portals of entry. Following, the ability of HO2, 223 and four additional isolates to invade a Chinook salmon embryo cell line, a fat head minnow cell line and a rainbow trout liver cell line was tested. All E. ictaluri isolates were invasive in all cell lines albeit at different degrees. Isolate HO2 was highly invasive in all cell lines with a significantly higher invasion capacity than isolate 223 in the Chinook salmon embryo cell line. A correlation between in vivo virulence and in vitro invasiveness hence is suggested although further studies are needed to confirm this hypothesis.     

Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.     

Inoculation of infectious pancreatic necrosis virus serotype Sp did not cause pancreas disease in Atlantic salmon (Salmo salar L.).

Atlantic salmon were selected from a fish farm with no previous record of pancreas disease (PD) or infectious pancreatic necrosis virus (IPNV) infection. Groups of fish were inoculated with either IPNV (strain Sp) from cell culture, organ material from fish with PD or control material as phosphate-buffered saline (PBS). Virological, histological and immunohistochemical examinations were carried out throughout the experiment. None of the fish died or showed clinical symptoms of PD. Histological examination revealed no pathological changes, and immunohistochemical studies were negative. Virus was isolated only sporadically from the group inoculated with organ material, whereas it was isolated consistently from the group inoculated with virus propagated in cell culture, as well as from in-contact control fish after the first week. In a latent carrier test, changes were entirely lacking in the first mentioned group, and were only slight in the last mentioned group. The data suggest that PD is not a transmissible disease, and that IPNV isolated from a PD outbreak does not play any part in the etiology of this disease.     

Clustering of parasites within cages on Scottish and Norwegian salmon farms: alternative sampling strategies illustrated using simulation

Within the literature, most discussion of sampling protocols for monitoring aquatic parasites is based on the assumptions of simple random sampling. Recent research has shown that in monitoring parasite abundance on fish farms composed of discrete cages, care must be taken to properly account for the clustering which naturally occurs. This paper illustrates the effect of clustering in the context of monitoring ectoparasitic sea lice Lepeophtheirus salmonis and Caligus elongatus in salmon farms. The degree of clustering of sea lice infections in fish within cages is measured using the intraclass correlation coefficient (ICC). A wide range of ICC values from sites in Scotland and Norway were estimated for the chalimus and mobile stages of L. salmonis, and for C. elongatus mobiles. The analyses indicate that significant clustering of lice infections within cages occurs across lice species and stages on both Scottish and Norwegian farms. A Monte-Carlo simulation using two sets of data from Scottish farms with ICC values for adult L. salmonis of 0.35 [0.08-0.73, 95% CI] and for adult C. elongatus of 0.39 [0.16-0.69, 95% CI] were used to illustrate the implications of clustering. The protocols simulated reflect those typically used across a range of countries and production environments in which salmon are currently reared. The findings demonstrate that the "few fish from many cages" approach results in a marked improvement in precision when sampling aquatic one-host parasites in cage-based production systems.     

Parasites in cultured and feral fish.

Parasites, causing little apparent damage in feral fish populations, may become causative agents of diseases of great importance in farmed fish, leading to pathological changes, decrease of fitness or reduction of the market value of fish. Despite considerable progress in fish parasitology in the last decades, major gaps still exist in the knowledge of taxonomy, biology, epizootiology and control of fish parasites, including such 'evergreens' as the ciliate Ichthyophthirius multifiliis, a causative agent of white spot disease, or proliferative kidney disease (PKD), one of the most economically damaging diseases in the rainbow trout industry which causative agent remain enigmatic. Besides long-recognized parasites, other potentially severe pathogens have appeared quite recently such as amphizoic amoebae, causative agents of amoebic gill disease (AGD), the monogenean Gyrodactylus salaris which has destroyed salmon populations in Norway, or sea lice, in particular Lepeophtheirus salmonis that endanger marine salmonids in some areas. Recent spreading of some parasites throughout the world (e.g. the cestode Bothriocephalus acheilognathi) has been facilitated through insufficient veterinary control during import of fish. Control of many important parasitic diseases is still far from being satisfactory and further research is needed. Use of chemotherapy has limitations and new effective, but environmentally safe drugs should be developed. A very promising area of future research seems to be studies on immunity in parasitic infections, use of molecular technology in diagnostics and development of new vaccines against the most pathogenic parasites.     

Sea lice infestations on farmed Atlantic salmon in Scotland and the use of ectoparasitic treatments

A recently compiled national database on sea lice infestations on farmed Atlantic salmon, contains detailed records for the period 1996 to 2000 from over 30 commercial sites on the west coast of Scotland. The data indicate that the two prevalent species of lice, Lepeophtheirus salmonis and Caligus elongatus, have different trends in abundance and distinctive seasonal patterns of infestation on farmed salmon. For the economically important species L salmonis, its abundance on fish varies with the time of the production cycle, the time of year and the particular year. Weekly fluctuations in sea lice counts indicate that treatment can be very effective in controlling infestations but that the counts recover rapidly and regular treatments are necessary to ensure control. A comparison of sites using medium or large numbers of treatments suggests that they do not reduce sea lice infestations to the same levels. There is also evidence that sites using treatments based on different chemical constituents had significantly different levels of infestation.     

Technical note: Modifying Atlantic salmon (Salmo salar) jumping behavior to facilitate innovation of parasitic sea lice control techniques

Industrial salmon farms are reservoirs of parasitic sea lice (Lepeophtheirus salmonis and Caligus spp.), which causes both production inefficiencies and contributes to population-level declines of wild salmon and trout. Current control methods vary in effect and stimulate controversy by the discharge of chemicals into the environment. An alternate control method uses a thin, chemical-infused oil layer on the sea surface. As farmed salmon jump through the surface, the treatment makes contact with the lipophilic carapace of sea lice and kills them. To enhance the effectiveness of this method, we tested whether the natural jumping behavior of salmon could be increased and directed. In a 2,000-m(3) experimental sea-cage, we removed the ability of groups of salmon to access the surface for different periods (0 to 48 h) and measured their surface behaviors after the surface became accessible again. Surface removal for 24 and 48 h induced 93% of salmon to jump in the 2 h after surface access was reinstated, a result that differed (P < 0.001) from the shorter duration (0 to 12 h) treatments. Salmon without surface access for 24 and 48 h jumped 2 to 3 times more often (P < 0.001), and made their first jump 2 to 3 times sooner (P = 0.003) on average after surface access became available than salmon in the shorter duration treatments. Our results indicate that removal of surface access for short periods may lead to loss of air from the physostomous swim bladder and cause negative buoyancy. This creates a behavioral drive for salmon to jump, swallow air and fill their swim bladders once surface access is reinstated. By combining the increased jumping behavior induced by this technique with a floating, oil-infused treatment, efficiency of sea lice treatments may be improved and treatment chemicals can be re-collected, thus decreasing environmental pollution.     

Glandular kallikrein in the innate immune system of Atlantic salmon (Salmo salar)

Glandular Kallikrein is a serine-protease with trypsin-like activity and is able to generate bioactive peptides from inactive precursors. We have evaluated the presence of this protease in the different organs of the Atlantic salmon (Salmo salar). The results clearly indicate that GK and PRL are generated in the same pituitary cells based on a co-localization by confocal microscopy. Based on probed cross-reactivity between C. striata and C. carpio glandular anti-GK antibodies, we used a homologous antibody to detect the presence of GK in several salmon tissues. We have evaluated the GK expression in healthy and defied fish. P. salmonis and V. ordalii. The GK immunoreaction in organs such as leukocytes, gills and skin is considerably increased in defied fish compared to healthy fish. This increase was present in the cells of the excretory kidney and in the intercellular tissue, where the development of hematopoietic and lymphocytic lines in fish take place. One of the most interesting organs to study was the skin, bearing in mind that this is a primary barrier to all pathogens. The skin of the defied fish exhibited an increase in immunoreactivity for glandular kallikrein similar to the protease found in mucus. An immunoreactive tissue kallikrein-like protein was identified and partially separated by perfusion chromatography. Enzymatic activity of salmon muscle prokallikrein was determined before and after trypsin activation. Kallikrein activity was characterized with respect to their ability to cleave the chromogenic leaving group, p-nitroanilide, from the peptidyl kallikrein and trypsin substrate. These findings constitute a important contribution to reveal the role of kallikrein in the innate immune system of fish.     

Evaluation of bithionol as a bath treatment for amoebic gill disease caused by Neoparamoeba spp

This study examined the toxicity of bithionol to Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss in fresh- and seawater and the efficacy of bithionol as a 1h seawater bath treatment for amoebic gill disease (AGD). To examine toxicity, fish were bathed for 1, 3 and 6h in bithionol, an anti-protozoal at 0, 1, 5, 10, 25 and 35mgL(-1) with toxicity determined by time to morbidity. Efficacy was examined by bathing AGD-affected Atlantic salmon and rainbow trout for 1h at bithionol concentrations of 1-25mgL(-1). Efficacy was determined by examining gill amoeba counts and identifying percent lesioned gill filaments at 1 and 24h after bath exposure to bithionol. For both species, bithionol was determined to be toxic at 25 and 35mgL(-1) exhibiting median lethal times (LT50s) ranging from 21 to 84min. Morbidity occurred in the 5 and 10mgL(-1) treatments, however, due to sampling regime there were not enough fish available to calculate LT50s. Only bithionol at 1mgL(-1) was considered non-toxic with no signs of morbidity. Bithionol was more toxic in seawater than freshwater and had no acute effects on gill Na+/K+ ATPase and succinic dehydrogenase, or plasma osmolality and chloride concentration. Bithionol at 1mgL(-1) reduced percent lesioned gill filaments in Atlantic salmon and rainbow trout by 33 and 27 per cent, respectively, compared to the seawater control. Similarly, numbers of amoeba were reduced by 33 and 43 per cent for Atlantic salmon and rainbow trout, respectively, when compared to the seawater control. Furthermore, bithionol reduced percent lesioned gill filaments as much as did the current industry standard of freshwater. This study demonstrated that a 1h seawater bath containing 1mgL(-1) bithionol could be an improvement to the current method of treatment for AGD-affected Atlantic salmon and rainbow trout.     

Correlation of virus replication in tissues with histologic lesions in Atlantic salmon experimentally infected with infectious salmon anemia virus

We have studied the replication of virus in tissues and development of lesions associated with infectious salmon anemia virus (ISAV) infection in Atlantic salmon using in situ hybridization (ISH) with a riboprobe targeting ISAV RNA segment 7 messenger RNA. Fish were infected with three ISAV isolates (U5575-1, RPC-01-0593-1, Norway 810/9/99) and then euthanatized sequentially at 3, 6, 10, and 13 days postinoculation (dpi) and thereafter once a week for 8 weeks. Severe histopathologic lesions were observed in tissues from all groups beginning at the onset of mortality. The severe histopathologic lesions correlated with maximum intensity and frequency of ISH signals (P < 0.001). There was a strong association between the hybridization signals and severity of lesions in the liver, kidney, and heart (R = 0.81, 0.70, and 0.78, respectively; P < 0.001). The distribution of ISH signals indicated the presence of a viremia because signals were observed predominantly in individual blood cells and endothelial cells, and possibly hematopoietic cells of head kidney, but not in the necrotic hepatocytes and renal epithelium. Of the organs sampled, the heart was the first and last to show ISH signals, possibly because of increased activity of the endocardial endothelial cells and the underlining macrophages, which continuously trap and remove circulating virus, and therefore represents the best tissue sample for screening of suspected infected fish. On the basis of mortality, severity of lesions, and intensity and frequency of ISH signals, ISAV isolate Norway 810/9/99 was the most virulent and U5575-1 the least virulent isolate studied.