Atlantic salmon paramyxovirus (ASPV) infection contributes to proliferative gill inflammation (PGI) in seawater-reared Salmo salar

Proliferative gill inflammation (PGI) causes significant losses in farmed Atlantic salmon Salmo salar L. in Norway, especially during the first months following seawater transfer. The aetiology is apparently multifactorial, including infection with chlamydia-like bacteria and Atlantic salmon paramyxovirus (ASPV). In the present study, gills from diseased fish from 3 farms on the western coast of Norway were sampled. The pathological changes were briefly described and the aetiological significance of ASPV studied by immunofluorescent staining of cryosections and by immunohistochemistry on sections of formalin-fixed and paraffin-embedded tissue. The pathological changes were macroscopically characterized by palour of the gills, and histologically by inflammation, circulatory disturbances, cell death and epithelial cell proliferation. ASPV was demonstrated in fish from all farms studied, as immunostaining consistent with ASPV was obtained in lamellar epithelial and endothelial cells of pathologically altered tissues. It is concluded that ASPV is at least a contributing cause of PGI. As far as we know, this is the first demonstration of fish disease related to infection with a paramyxovirus.     

A novel betaproteobacterial agent of gill epitheliocystis in seawater farmed Atlantic salmon (Salmo salar)

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although 'Candidatus Piscichlamydia salmonis', previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as 'Candidatus Branchiomonas cysticola', was the cyst-forming agent in these samples. While histology and ultrastructure of 'Ca. B. cysticola' cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.     

Cranial nodules in farmed Atlantic salmon, Salmo salar L., fry

Cranial nodules are described from Atlantic salmon, Salmo salar fry hatched from certain egg batches. Conspicuous, smooth, mostly unilateral nodules were recorded on the cranium. Histologically, the cerebellum appeared normal, but was displaced dorsally. Karyorhectic malpighian cells were absent from the epidermis and the meninges appeared normal. There was no apparent contact of the cerebellum with the water, or evidence of fungal, bacterial or parasitic infection. Mortality of up to 15% as recorded. The aetiology of this condition is currently unknown.     

An epidemiological study of cataracts in seawater farmed Atlantic salmon Salmo salar

Cataracts in farmed Atlantic salmon have been known for many years, but the aetiology and importance of the disease have not been clarified. A cross-sectional field study of 51 cages of Atlantic salmon at 49 randomly selected sea sites was performed during the summer of 1998. The target population was spring and autumn entry groups of the 1997 generation salmon. Approximately 15 fish from each cage, altogether 777 fish, were autopsied by the same person. Each eye of the fish was scored for cataracts on a scale from 0 to 4 using an otoscope lamp with magnification. The weight and length of each fish were measured. The prevalence of cataracts was 83 % and 79% in spring entry groups and autumn entry groups, respectively. The overall mean cataract index (mean score of both eyes) was 1.23, being significantly higher in the spring entry groups (1.36) than the autumn entry groups (0.85). The final results in the spring entry groups showed that the fish groups with higher weight at sea transfer also had a higher cataract index at inspection. The risk of development of cataracts varied significantly among the offspring from the 5 strains represented in the study. Fish from sites located in 2 counties in the southern part of Norway had a significantly higher cataract index than fish farmed in the northernmost county in the study. For the autumn entry groups none of the explanatory variables was significant. In the spring entry groups a significant negative relationship was observed between the cataract score and the weight of the fish at the time of inspection (Pearson's r = -0.17), while the corresponding correlation for the autumn released groups was r = -0.10. Among the spring entry groups the average weight of the fish with the highest cataract score was estimated to about a third of the weight of the fish with no visible cataracts.     

Characterisation of an emerging rickettsia-like organism in Tasmanian farmed Atlantic salmon Salmo salar

A rickettsia-like organism (RLO) was observed in farmed Atlantic salmon Salmo salar located in south-east Tasmania, Australia. Several assays such as immunoperoxidase, immunoelectron microscopy, polymerase chain reaction and nucleic acid sequencing, as well as phylogenetic analysis of rDNA sequences, were performed on infected fish tissues. Immunohistochemistry results suggested the presence of related antigenic determinants between the Tasmanian RLO and the type strain LF-89 of Piscirickettsia salmonis. However, sequence alignment demonstrated that the Tasmanian RLO contains a 19 bp deletion at the 3'-end of the internal transcribed spacer region of the rDNA operon, indicating a genetic divergence from P. salmonis isolates, which are exotic to Australia.     

Spawning behaviour of a farmed escaped female Atlantic salmon (Salmo salar)

Excavation of stranded redds revealed differences in spawning behaviour between farmed and wild Atlantic salmon. The redd of a farmed fish contained more egg pockets (nine v . average of two) and fewer eggs per pocket (averages: 459 v . 707). No other pocket measures differed.     

Migratory behaviour of wild and farmed Atlantic salmon (Salmo salar) during spawning

Migratory behaviour at spawning of wild and newly-escaped farmed Atlantic salmon was analysed by radio telemetry in the River Alta, North Norway. Spawning areas were located by aerial surveys. Farmed females moved significantly more than wild females ( P <0.01). There was no such difference between the two groups of males. About 83% of the wild fish stayed within identified spawning areas for 1 day or longer. The corresponding figure for farmed salmon was only 43% ( P <0.05). Wild salmon stayed 8.1 days inside spawning areas and farmed salmon 5.2 days. The present results suggest that escaped farmed salmon had reduced spawning success compared with wild fish.     

Heart morphology in wild and farmed Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss

The normal shape of the salmonid ventricle is a triangular pyramid with the apex pointing caudoventrally. A strong positive correlation has been established between this shape and optimum cardiac output and function. Domesticated salmonids appear to have developed a more rounded ventricle with misaligned bulbus arteriosus. Several reports from fish health veterinarians indicate that fish with abnormal heart morphology have a high mortality rate during stress-inducing situations like grading, transportation and bath treatments. The present paper compares and describes the ventricle morphology of wild vs. farmed Atlantic salmon, and wild steelhead (anadromous rainbow trout) vs. farmed rainbow trout. Several parameters were measured to provide numerical measurement of the differences in shape, i.e. height:width ratio and the angle between the longitudinal ventricular axis and the axis of the bulbus arteriosus. We conclude that the hearts of farmed fish are rounder than those in corresponding wild fish, and that the angle between the ventricular axis and the axis of the bulbus arteriosus is more acute in wild fish than in their farmed counterparts. Further studies are necessary to reveal the prevalence, functional significance and possible causes of these abnormal hearts.     

Diseases of farmed Atlantic salmon Salmo salar associated with infections by the microsporidian Paranucleospora theridion

The microsporidian Paranucleospora theridion was discovered in Atlantic salmon Salmo salar suffering from proliferative gill disease in a marine farm in western Norway in 2008. The parasite develops in cells of the reticuloendothelial system, cells important for normal immune function. The aim of this study was to see if P. theridion could play a part in some of the diseases with unclear causes in salmon production in Norway, i.e. proliferative gill disease (PGI), pancreas disease (PD), heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS). P. theridion was present in all areas with salmon farming in Norway, but high prevalence and densities of the parasite in salmon and salmon lice were only seen in southern Norway. This region is also the main area for PGI and PD in Norway. Quantification of pathogens associated with PGI, PD, HSMI and CMS diagnoses showed that P. theridion levels are high in southern Norway, and may therefore play a role in susceptibility and disease development. However, among the different diagnoses, fish with PGI are particularly heavily infected with P. theridion. Therefore, P. theridion appears as a possible primary agent in cases with high mortality in connection with PGI in western Norway.     

Normal ranges of some blood chemistry parameters in adult farmed Atlantic salmon, Salmo salar

Blood samples from healthy adult Atlantic salmon fed an optimal diet in net sea pens were collected at intervals from October to May. Haematological determinations and biochemical serum analyses were carried out on 20 fish in each of seven samples. The ranges of haemato-logical values for sample means were: haematocrit 44–49%, haemoglobin 8.9–10.4 g 100ml⁻¹, red blood cell count 0.85–1.10 × 10¹² l⁻¹, MCV 441–553 × 10⁻¹⁵ 1, MCH 94–106 × 10⁻⁶ g, MCHC 19.4–21.7 g 100ml⁻¹ and leucocrit 0.43–0.96%. The ranges of enzyme activities in serum, for sample means, were: alkaline phosphatase 647–988Ul⁻¹, aspartate aminotrans-ferase 202–351 Ul⁻¹ and alanine aminotransferase 4–8 Ul⁻¹. The ranges of the other parameters analyzed in serum were: total protein 41.6–56.6 gl⁻¹, albumin 18.3–24.3 gl⁻¹, albumin/total protein ratio 39.3–44.0%, creatinine 26–46 μmol, triglycerides 2.53–4.98 μmol and cholesterol 9.3–12.8 μmol. These values are considered to be the normal ranges in healthy fish. Variations due to seasonal changes, and the clinical significance of the selected parameters, are discussed. Data showing the reproducibility of the biochemical analyses in serum are presented.     

Molecular characterization of the myxosporean associated with parasitic encephalitis of farmed Atlantic salmon Salmo salar in Ireland

During seasonal epizootics of neurologic disease and mass mortality in the summers of 1992, 1993 and 1994 on a sea-farm in Ireland, Atlantic salmon Salmo salar smolts suffered from encephalitis associated with infection by a neurotropic parasite. Based on ultrastructural studies, this neurotropic parasite was identified as an intercellular presporogonic multicellular developmental stage of a histozoic myxosporean, possibly a Myxobolus species. In order to generate sequence data for phylogenetic comparisons to substantiate the present morphological identification of this myxosporean in the absence of detectable sporogony, polymerase chain reaction (PCR), Southern blot hybridization, dideoxynucleotide chain-termination DNA sequencing, and in situ hybridization (ISH) were used in concert to characterize segments of the small subunit ribosomal RNA (SSU rRNA) gene. Oligonucleotide primers were created from sequences of the SSU rRNA gene of M. cerebralis and were employed in PCR experiments using DNA extracted from formalin-fixed paraffin-embedded tissue sections of brains from Atlantic salmon smolts in which the myxosporean had been detected by light microscopy. Five segments of the SSU rRNA gene of the myxosporean, ranging in length from 187 to 287 base pairs, were amplified, detected by hybridization with sequence-specific probes, and sequenced. Consensus sequences from these segments were aligned to create a partial sequence of the SSU rRNA gene of the myxosporean. Assessments of sequence identity were made between this partial sequence and sequences of SSU rRNA genes from 7 myxosporeans, including Ceratomyxa shasta, Henneguya doori, M. arcticus, M. cerebralis, M. insidiosus, M. neurobius, and M. squamalis. The partial SSU rRNA gene sequence from the myxosporean had more sequence identity with SSU rRNA gene sequences from neurotropic and myotropic species of Myxobolus than to those from epitheliotropic species of Myxobolus or Henneguya, or the enterotropic species of Ceratomyxa, and was identical to regions of the SSU rRNA gene of M. cerebralis. Digoxigenin-labeled oligonucleotide DNA probes complementary to multiple segments of the SSU rRNA gene of M. cerebralis hybridized with DNA of the parasite in histologic sections of brain in ISH experiments, demonstrating definitively that the segments of genome amplified were from the organisms identified by histology and ultrastructural analysis. Based on sequence data derived entirely from genetic material of extrasporogonic stages, the SSU rDNA sequence identity discovered in this study supports the hypothesis that the myxosporean associated with encephalitis of farmed Atlantic salmon smolts is a neurotropic species of the genus Myxobolus, with sequences identical to those of M. cerebralis.     

Aspects of the epizootiology of pancreas disease in farmed Atlantic salmon Salmo salar in Ireland.

A computerised database containing information on over 17.8 million salmon contained within 49 separate marine populations was used to study the epidemiology of pancreas disease (PD) in Ireland. Of the 43 recorded PD outbreaks, 57% occurred in the 3 mo period August to October inclusive (17 to 32 wk post-transfer). Analysis of variance of mortality rates during PD outbreaks occurring on 6 marine sites over a 5 yr period showed that mortality rates vary significantly between sites (p < 0.001) but not between years over this time period. The mortality rate during PD outbreaks ranged from 0.1 to 63%. Mortality rates were significantly higher when PD outbreaks occurred earlier in the year (y = -1.28x + 59, SE of b 0.33). The mean length of a PD outbreak was 112 d (SE = 7.7, n = 37). There was no correlation between PD mortality rate and smolt input weight, initial stocking density and transfer mortality.     

Cardiac pathological changes of Atlantic salmon (Salmo salar L.) affected with heart and skeletal muscle inflammation (HSMI)

Heart and skeletal muscle inflammation (HSMI) is a disease of marine farmed Atlantic salmon where the pathological changes associated with the disease involve necrosis and an infiltration of inflammatory cells into different regions of the heart and skeletal muscle. The aim of this work was to characterize cardiac changes and inflammatory cell types associated with a clinical HSMI outbreak in Atlantic salmon using immunohistochemistry. Different immune cells and cardiac tissue responses associated with the disease were identified using different markers. The spectrum of inflammatory cells associated with the cardiac pathology consisted of mainly CD3(+) T lymphocytes, moderate numbers of macrophages and eosinophilic granulocytes. Proliferative cell nuclear antigen (PCNA) immuno-reaction identified significantly increased nuclear and cytoplasmic staining as well as identifying hypertrophic nuclei. Strong immunostaining was observed for major histocompatibility complex (MHC) class II in HSMI hearts. Although low in number, a few positive cells in diseased hearts were detected using the mature myeloid cell line granulocytes/monocytes antibody indicating more positive cells in diseased than non-diseased hearts. The recombinant tumor necrosis factor-α (TNFα) antibody identified stained macrophage-like cells and endothelial cells around lesions in addition to eosinophilic granular cells (EGCs). These findings suggested that the inflammatory response in diseased hearts comprised of mostly CD3(+) T lymphocytes and eosinophilic granular cells and hearts exhibited high cell turnover where DNA damage/repair might be the case (as identified by PCNA, caspase 3 and terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) reactivity).     

Respiratory pathogenesis of amoebic gill disease (AGD) in experimentally infected Atlantic salmon Salmo salar

The aim of this study was to investigate the respiratory responses of Atlantic salmon, Salmo salar, experimentally affected with amoebic gill disease (AGD). In Series I, arterial blood samples were taken over a 96 h period following amoebae addition to examine potential respiratory effects associated with initial exposure. No major significant treatment effects were found between fish exposed to amoebae and control (non-exposed) fish. Arterial pH (pHa) was seen to be significantly elevated at 48 h in AGD fish relative to the 0 h time point. To investigate the long-term respiratory effects associated with infection, fish were similarly exposed to amoebae and sampled over a 16 d period. As for Series I, caudal blood pH was significantly elevated by Day 2 (48 h) compared to the pre (Day 0)-time point, suggesting that initial exposure to amoebae and/or amoebae attachment may have induced an initial respiratory alkalosis via increased ventilation frequency and/or amplitude. From Day 7 onwards, and coinciding with a significant increase in the percentage of affected gill filaments, blood pH decreased significantly, possibly indicating the onset of the characteristic respiratory acidosis that has previously been described for experimentally AGD-affected Atlantic salmon. Although fish in this study showed up to 90% AGD-affected filaments, the corresponding respiratory results do not reflect a major acid-base disturbance. Therefore, the findings from the present study support the contention that, although AGD only affects the gill, AGD-associated mortality in Atlantic salmon may not be primarily associated with respiratory failure.     

Serological and molecular heterogeneity among Yersinia ruckeri strains isolated from farmed Atlantic salmon Salmo salar in Chile

We investigated 11 strains of Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), that had been isolated from Atlantic salmon Salmo salar L. farmed in Chile and previously vaccinated against ERM. Phylogenetic analysis of the 16S rRNA gene sequences confirmed the identification of the salmon isolates as Y. ruckeri. A comparative analysis of the biochemical characteristics was made by means of traditional and commercial miniaturised methods. All studied isolates were motile and Tween 80 positive, and were identified as biotype 1. In addition, drug susceptibility tests determined high sensitivity to sulphamethoxazole/trimethroprim, oxytetracycline, ampicillin and enrofloxacin in all isolates. Serological assays showed the presence of O1a, O1b and O2b serotypes, with a predominance of the O1b serotype in 9 strains. Analysis of the lipopolysaccharide profiles and the correspondent immunoblot confirmed these results. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of the outer membrane proteins revealed that all Chilean strains had profiles with a molecular weight range between 34 and 55 kDa, with 3 distinct groups based on differences in the major bands. Genotyping analyses by enterobacterial repetitive intergenic consensus (ERIC-) and repetitive extragenic palindromic (REP-)PCR techniques clearly indicated intraspecific genetic diversity among Chilean Y. ruckeri strains.     

Major histocompatibility polymorphism associated with resistance towards amoebic gill disease in Atlantic salmon (Salmo salar L.)

The association between major histocompatibility (MH) polymorphism and the severity of infection by amoebic gill disease (AGD) was investigated across 30 full sibling families of Atlantic salmon. Individuals were challenged with AGD for 19days and then their severity of infection scored by histopathological examination of the gills. Fish were then genotyped for the MH class I (Sasa-UBA) and MH class II alpha (Sasa-DAA) genes using polymorphic repeats embedded within the 3' untranslated regions of the Sasa-UBA and Sasa-DAA genes. High variation in the severity of infection was observed across the sample material, ranging from 0% to 85% gill filaments infected. In total, seven Sasa-DAA-3UTR and ten Sasa-UBA-3UTR marker alleles were identified across the 30 families. A significant association between the marker allele Sasa-DAA-3UTR 239 and a reduction in AGD severity was detected. There was also a significant association found between AGD severity and the presence of two Sasa-DAA-3UTR genotypes. While the associations between MH allele/genotypes and AGD severity reported herein may be statistically significant, the small sample sizes observed for some alleles and genotypes means these associations should be considered as suggestive and future research is required to verify their biological significance.     

Gonadotropic cells in the Atlantic salmon,Salmo salar

Summary The gonadotropin-producing cells (GTH-cells) in the Atlantic salmon were studied light and electron microscopically before, during and after spawning, and after injections of luteinizing hormone-releasing hormone (LH-RH). The double immunofluorescent technique was applied using rabbit anti-carp GTH as the first antibody. Numerous immunofluorescent cells were observed throughout the pars distalis, but very few in the pars intermedia. These cells are basophilic and PAS-positive, and ultrastructurally classified as globular gonadotropes. Only one gonadotropic cell type could be identified; its size, morphology and fine structure vary considerably. In the same specimen the GTH-cells can be predominantly globular or vesicular in appearance, depending on the reproductive phase of the fish. At spawning and after LH-RH injection, many GTH-cells reach a vacuolar stage; the content of the vacuoles is not immunofluorescent. Another cell type, which resembles GTH-cells in semithin sections, did not show gonadotropic properties; its nature and functional significance are unknown. In addition, the present study revealed an increase in the synthetic and exocytotic activity of prolactin cells after LH-RH injections. It is suggested that LH-RH mediates this effect via LH and eventually via estradiol.The authors are indebted to Dr. N. Johansson and superintendent N. Steffner for generously providing facilities at their institutes in Älvkarleby. They also wish to thank Mrs. Y. Lilliemarck and Mrs. V. Lundin for their skillful technical assistance. The grants made available by the Swedish Natural Science Research Council (No. 2124-037) are also gratefully acknowledged. Thanks are also due to Mrs. S.M. McNab for linguistic assistance