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1.
Formaldehyde‐based fixatives are generally employed in histopathology despite some significant disadvantages associated with their usage. Formaldehyde fixes tissue by covalently cross‐linking proteins, a process known to mask epitopes which in turn can reduce the intensity of immunohistochemical stains widely used in disease diagnostics. Additionally, formaldehyde fixation greatly limits the ability to recover DNA and mRNA from fixed specimens to the detriment of further downstream molecular analyses. Amoebic gill disease (AGD) has been reliably diagnosed from histological examination of gills although complementary methods such as in situ hybridization (ISH) and polymerase chain reaction (PCR) are required to confirm the presence of Neoparamoeba perurans, the causative agent of AGD. As molecular techniques are becoming more prevalent for pathogen identification, there is a need to adapt specimen collection and preservation so that both histology and molecular biology can be used to diagnose the same sample. This study used a general approach to evaluate five different fixatives for Atlantic salmon, Salmo salar L., gills. Neutral‐buffered formalin and seawater Davidson's, formaldehyde‐based fixatives commonly used in fish histopathology, were compared to formalin‐free commercial fixatives PAXgene®, HistoChoice?MB* and RNAlater?. Each fixative was assessed by a suite of analyses used to demonstrate AGD including routine histochemical stains, immunohistochemical stains, ISH and DNA extraction followed by PCR. All five fixatives were suitable for histological examination of Atlantic salmon gills, with seawater Davidson's providing the best quality histopathology results. Of the fixatives evaluated seawater Davidson's and PAXgene® were shown to be the most compatible with molecular biology techniques. They both provided good DNA recovery, quantity and integrity, from fixed and embedded specimens. The capacity to preserve tissue and cellular morphology in addition to allowing molecular analyses of the same specimens makes seawater Davidson's and PAXgene® appear to be the best fixation methods for diagnosis and research on AGD in Atlantic salmon gills.  相似文献   

2.
There is inconsistent evidence of resistance of Atlantic salmon, Salmo salar L., to amoebic gill disease (AGD). Here, evidence is presented that demonstrates that Atlantic salmon exposed and subsequently challenged with AGD are more resistant than naïve control fish. Seventy‐three per cent of Atlantic salmon previously exposed to AGD survived to day 35 post‐challenge compared with 26% exposed to Neoparamoeba sp. for the first time, yet the gill pathology of surviving naïve control or previously exposed fish was not significantly different. Development of resistance to AGD is associated with anti‐Neoparamoeba sp. antibodies that were detectable in serum of 50% of surviving Atlantic salmon previously exposed to AGD. However, anti‐Neoparamoeba sp. antibodies were not detectable in cutaneous mucus of resistant fish. Increased resistance of Atlantic salmon after secondary Neoparamoeba sp. infection and detection of specific serum antibodies provides support for the development of a vaccine for AGD.  相似文献   

3.
Fish gills are heavily exposed to the external milieu and may react against irritants with different cellular responses. We describe variations in mucous cell counts in gills from healthy Atlantic salmon (Salmo salar) presmolts in five recirculating aquaculture system (RAS) farms and one flow‐through farm. Based on certain criteria, mucous cells were histologically quantified in a defined lamellar region of the gills and the counts were analysed. Immunohistochemistry (IHC) was used to investigate epithelial responses. The median number of total mucous cells in the defined region was 59 per fish. Between the farms, the medians varied from 31 to 101 with the lowest in the flow‐through farm. A regression model was fitted with “total mucous cells” as the dependent variable and with “fish length” and “fish farm” as independent variables. The proportion of variation in mucous cell counts explained by the model was twice as high when “fish farm” was included compared to only “fish length.” IHC revealed proliferative responses in coherence with high mucous cell numbers. Conclusively, the variation in mucous cell counts depends on combined farm‐related factors. Establishing a baseline for mucous cell counts is fundamental in the development of high‐throughput monitoring programmes of gill health in farmed fish.  相似文献   

4.
Previous work in our laboratory defined a method of inducing laboratory‐based amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L. Gills of AGD‐affected fish were scraped and the debris placed into fish‐holding systems, eliciting AGD in naïve Atlantic salmon. While this method is consistently successful in inducing AGD, variability in the kinetics and severity of infections has been observed. It is believed that the infections are influenced by inherently variable viability of post‐harvest amoeba trophozoites. Here, a new method of experimental induction of AGD is presented that redefines the infection model including the minimum infective dose. Amoebae were partially purified from the gills of AGD‐affected Atlantic salmon. Trophozoites were characterized by light microscopy and immunocytochemistry and designated Neoparamoeba sp., possibly Neoparamoeba pemaquidensis. Cells were placed into experimental infection systems ranging in concentration from 0 to 500 cells L?1. AGD was detected by gross and histological examination in fish held in all systems inoculated with amoebae. The number of gross and histological AGD lesions per gill was proportional to the inoculating concentration of amoebae indicating that the severity of disease is a function of amoeba density in the water column. The implications of these observations are discussed in the context of the existing AGD literature base as well as Atlantic salmon farming in south‐eastern Tasmania.  相似文献   

5.
A 2-year study was carried out on amoebic gill disease (AGD) involving monthly samples of 1+ Atlantic salmon, Salmo salar L., smolts, histological assessment of the gills and analysis of environmental data. Gill pathology was seen before amoebae could be detected microscopically. These changes in gill integrity were associated with marine environmental conditions, particularly elevated ammonium, nitrite and chlorophyll levels. The results suggest that the environmental changes predispose salmon to colonization by amoebae and ciliates. High densities of histophagous scuticociliates were observed in the gills during periods of advanced gill pathology. A number of different amoebae were observed in close association with gill pathology. Neoparamoeba was not seen in high densities, nor was it associated with gill pathology, indicating that Neoparamoeba may not be the primary agent of the AGD in Irish salmonid culture.  相似文献   

6.
The treatment of amoebic gill disease (AGD) in cultured Atlantic salmon, Salmo salar L., using mucolytic agents has been previously reported. The agent L‐cysteine ethyl ester reduces salmonid mucus viscosity and potentially increases the flushing of the gill. In the present study, the effects of the mucolytic agent N‐acetyl cysteine (NAC) were assessed. Cutaneous mucus from rainbow trout, Oncorhynchus mykiss Walbaum, and Atlantic salmon was shown to have reduced viscosity when mixed in vitro with 100 or 200 μg/mL NAC. Saltwater‐acclimated rainbow trout and Atlantic salmon were fed an oil‐incorporated, NAC‐medicated diet (8 g NAC/kg diet) for up to 24 d and challenged with inoculation of 300 cells/L Neoparamoeba spp., the etiological agent of AGD. Control fish were fed normal oil‐coated pellets and received no NAC. NAC medication failed to reduce the severity of gill lesions associated with AGD even though the mucus viscosity from medicated fish was less than that of controls. Oral NAC medication does not appear to be an effective method for controlling AGD in salmonids despite reducing cutaneous mucus viscosity.  相似文献   

7.
A relationship between increasing water temperature and amoebic gill disease (AGD) prevalence in Atlantic salmon (Salmo salar) has been noted at fish farms in numerous countries. In Scotland (UK), temperatures above 12°C are considered to be an important risk factor for AGD outbreaks. Thus, the purpose of this study was to test for the presence of an association between temperature and variation in the severity of AGD in Atlantic salmon at 10 and 15°C. The results showed an association between temperature and variation in AGD severity in salmon from analysis of histopathology and Paramoeba perurans load, reflecting an earlier and stronger infection post‐amoebae exposure at the higher temperature. While no significant difference between the two temperature treatment groups was found in plasma cortisol levels, both glucose and lactate levels increased when gill pathology was evident at both temperatures. Expression analysis of immune‐ and stress‐related genes showed more modulation in gills than in head kidney, revealing an organ‐specific response and an interplay between temperature and infection. In conclusion, temperature may not only affect the host response, but perhaps also favour higher attachment/growth capacity of the amoebae as seen with the earlier and stronger P. perurans infection at 15°C.  相似文献   

8.
Amoebic gill disease (AGD) caused by the ectoparasite Paramoeba perurans affects several cultured marine fish species worldwide. In this study, the morphology and ultrastructure of P. perurans in vitro and in vivo was investigated using scanning and transmission electron microscopy (SEM and TEM, respectively). Amoebae cultures contained several different morphologies ranging from a distinct rounded cell structure and polymorphic cells with pseudopodia of different lengths and shapes. SEM studies of the gills of AGD‐affected Atlantic salmon, Salmo salar L., revealed the presence of enlarged swellings in affected gill filaments and fusion of adjacent lamellae. Spherical amoebae appeared to embed within the epithelium, and subsequently leave hemispherical indentations with visible fenestrations in the basolateral surface following their departure. These fenestrated structures corresponded to the presence of pseudopodia which could be seen by TEM to penetrate into the epithelium. The membrane–membrane interface contained an amorphous and slightly fibrous matrix. This suggests the existence of cellular glycocalyces and a role for extracellular products in mediating pathological changes in amoebic gill disease.  相似文献   

9.
A study of microfauna, associated with pathological changes in the gills of Atlantic salmon, Salmo salar L., was conducted over 2001-2002. Monthly samples of 1(+) salmon smolts were taken, protozoan populations were quantified and gill health was assessed histologically. Protozoan densities were correlated with pathological changes, in order to determine their possible role in lesions in the gills. The most severe gill tissue changes were observed in summer/autumn and the least in spring. A diverse polyphyletic protozoan community was observed colonizing the gills, including Neoparamoeba sp., other amoebae, scuticociliates, Ichthyobodo-like flagellates, trichodinid ciliates and prostomatean ciliates. The earlier gill tissue changes in the gill were not always associated with the presence of these microorganisms, whereas amoebae (other than Neoparamoeba sp.), Ichthyobodo-like flagellates and trichodinid ciliates correlated with augmenting gill lesions. Neoparamoeba sp. was present, but its abundance did not correlate with the disease. This study suggests that a diversity of protozoans including Ichthyobodo-like flagellates, trichodinid ciliates and amoebae other than Neoparamoeba sp. are involved in the aetiology of amoebic gill disease in the Irish situation.  相似文献   

10.
Freshwater bathing is essential for control of amoebic gill disease (AGD) during the marine phase of the Tasmanian Atlantic salmon production cycle, a practice that is costly, production limiting and increasing in frequency. Although the pathogenesis of gill infection with Neoparamoeba sp. in naïve Atlantic salmon, Salmo salar, is now understood, the progression of re‐infection (post‐treatment) required elucidation. Here, we describe the weekly histopathological progression of AGD from first to second freshwater bath. Halocline cessation and increased water temperature appeared to drive the rapid onset of initial infection prior to bathing. Freshwater bathing cleared lesions of attached trophozoites and associated cellular debris. Subsequent gill re‐infection with Neoparamoeba sp. was evident at 2 weeks post‐bath and had significantly increased (P < 0.001), in severity by 4 weeks post‐bath. No significant difference in gross pathology was observed until 4 weeks post‐bath (P < 0.05). The re‐infective progression of AGD was characterized by localized host tissue responses juxtaposed to adhered trophozoites (epithelial oedema, hypertrophy and hyperplasia), non‐specific inflammatory cell infiltration (macrophages, neutrophils and eosinophilic granule cells) and finally advanced hyperplasia with epithelial fortification. During the post‐bath period, non‐AGD lesions including haemorrhage, necrosis and regenerative hyperplasia were occasionally observed, although no evidence of secondary colonization of these lesions by Neoparamoeba sp. was noted. We conclude that pathogenesis during the inter‐bath period was identical to initial infection although the source of re‐infection remains to be established.  相似文献   

11.
Atlantic salmon with amoebic gill disease (AGD) were treated with chloramine‐T to compare its effectiveness with that of freshwater bathing. In 250‐L tank trials, treatment of seawater with chloramine‐T reduced amoeba density on the gills to levels significantly lower than when treated with seawater alone. There was no further change in amoeba levels in fish bathed for 3 or 6 h compared with 1 h of treatment. Plasma lactate levels in fish bathed in chloramine‐T for 6 h showed no differences across treatments. In 1000‐L tank trials using freshwater alone or seawater with chloramine‐T, significant reductions in amoeba density occurred compared with pre‐bath levels. Histological analysis of gill tissue revealed AGD lesion levels to increase, then to return to pre‐bath levels within 1 week for freshwater‐treated fish, while chloramine‐T‐ and seawater‐treated fish had higher levels of AGD lesions from 2 weeks post bathing. Immunodot‐blot data indicated an initial significant increase in prevalence of lesions in seawater and chloramine‐T‐treated fish, which declined to levels significantly lower than pre‐bath levels by 3 weeks post bathing, compared with the freshwater‐treated fish, which had significantly lower levels than controls by 2 weeks post bathing. At reducing amoeba density, it is apparent that bathing AGD‐affected Atlantic salmon in seawater with chloramine‐T proved at least as effective as freshwater.  相似文献   

12.
Gill diseases cause considerable losses in Norwegian salmon farming. In 2015, we characterized salmon gill poxvirus (SGPV) and associated gill disease. Using newly developed diagnostic tools, we show here that SGPV infection is more widely distributed than previously assumed. We present seven cases of complex gill disease in Atlantic salmon farmed in seawater and freshwater from different parts of Norway. Apoptosis, the hallmark of acute SGPV infection, was not easily observed in these cases, and qPCR analysis was critical for identification of the presence of SGPV. Several other agents including Costia‐like parasites, gill amoebas, Saprolegnia spp. and bacteria were observed. The studied populations experienced significant mortalities, which increased to extreme levels when severe SGPV infections coincided with smoltification. SGPV infection appears to affect the smoltification process directly by affecting the gills and chloride cells in particular. SGPV may be considered a primary pathogen as it was often found prior to identification of complex gill disease. It is hypothesized that SGPV‐induced gill damage may impair innate immunity and allow invasion of secondary invaders. The distinct possibility that SGPV has been widely overlooked as a primary pathogen calls for extended use of SGPV qPCR in Atlantic salmon gill health management.  相似文献   

13.
This study surveyed conditions in the gills of wild marine fish in Tasmania to determine potential interactions between wild and cultured fish. Wild marine fish of 12 species were captured from three Atlantic salmon farm sites and three reference sites around Tasmania. The survey concentrated on three species, red cod, Pseudophycis bachus, sand flathead, Platycephalus bassensis, and jack mackerel, Trachurus declivus. Seventy-six per cent of salmon pens contained wild fish species. The number of species found in a pen ranged from one to nine and the number of individuals ranged from one to 23. Trichodinids were prevalent and occurred on seven of the 13 species examined. Trichodina occurred on the gills of all but one specimen of red cod. Monogenean gill flukes were observed on all three major species sampled and were abundant on sand flathead. Other parasites and conditions observed in the survey included metacercariae of digenean trematodes, epitheliocystis and cysts of unknown origin. Infestations of trichodinids on red cod and monogenean gill flukes on sand flathead were significantly more intense at farm sites than at reference sites. Atlantic salmon sampled at the same time from the farms were only affected by amoebic gill disease and isopods.  相似文献   

14.
Gill diseases are a complex and multifactorial challenge for marine farmed Atlantic salmon. Co‐infections with putative pathogens are common on farms; however, there is a lack of knowledge in relation to the potential effect co‐infections may have on pathology. The objective of this study was to determine the prevalence and potential effects of Neoparamoeba perurans, Desmozoon lepeophtherii, Candidatus Branchiomonas cysticola, Tenacibaculum maritimum and salmon gill poxvirus (SGPV) during a longitudinal study on a marine Atlantic salmon farm. Real‐time PCR was used to determine the presence and sequential infection patterns of these pathogens on gill samples collected from stocking until harvest. A number of multilevel models were used to determine the effect of these putative pathogens on gill health (measured as gill histopathology score), while adjusting for the effect of water temperature and time since the last freshwater treatment. Results indicate that between 12 and 16 weeks post‐seawater transfer (wpst), colonization of the gills by all pathogens had commenced and by week 16 of marine production each of the pathogens had been detected. D. lepeophtherii and Candidatus B. cysticola were by far the most prevalent of the potential pathogens detected during this study. Detections of T. maritimum were found to be significantly correlated with temperature showing distinct seasonality. Salmon gill poxvirus was found to be highly sporadic and detected in the first sampling point, suggesting a carryover from the freshwater stage of production. Finally, the model results indicated no clear effect between any of the pathogens. Additionally, the models showed that the only variable which had a consistent effect on the histology score was N. perurans.  相似文献   

15.
Atlantic salmon were exposed to amoebic gill disease (AGD) immediately following their acclimatization to sea water (group 1), or following a 2 week period of maintenance in sea water (group 2). Three fish from each group were sampled on days 0, 1, 2, 4, 7, 14 and 28 post-infection. Characteristic gill lesions began to occur between days 2 and 4, and dramatically increased by day 7. The number of gill lesions on fish from group 2 was significantly higher than on fish from group 1 on days 7 and 14 ( P <0.001), but the two groups did not differ in any other parameter. Histologically, Paramoeba sp., the aetiological agent of AGD, could be seen on the gills of fish as soon as 1 day post-exposure, attached to healthy-appearing gills. Gill pathology in the form of hyperplasia and lamellar fusion followed shortly. AGD infection was accompanied by a significant increase in the number of gill mucous cells ( P =0.002). Different methods for the diagnosis of AGD are discussed.  相似文献   

16.
Previous studies have indicated that when Atlantic salmon, Salmo salar L., are exposed to Neoparamoeba sp. the fish produce anti-Neoparamoeba sp. antibodies. It appears unlikely that these antibodies elicit any specific protection against amoebic gill disease (AGD) as fish with demonstrable activities have been affected by AGD. Experiments were conducted on Atlantic salmon cultured throughout Tasmania to assess the natural production of antibodies towards Neoparamoeba sp. Fish were sampled from areas where AGD was prevalent and from areas where there had been no reported cases. An enzyme-linked immunosorbent assay (ELISA) was used to measure anti-Neoparamoeba sp. antibody activities in serum. All fish from sea water had antibody activities greater than the negative control fish, including fish from areas with no reported cases of AGD. Time trial samples indicated that time after transfer to sea water did not appear to be a significant (P > 0.05) factor in antibody activity, however location was (P < 0.05). There was no agreement (corrected kappa value, 0.16) between the ELISA result and the isolation of Neoparamoeba sp. from the gills of the same fish. The results suggest that Atlantic salmon in seawater culture in Tasmania produce anti-Neoparamoeba sp. antibodies regardless of infection history, suggesting the presence of Neoparamoeba sp. in the environment.  相似文献   

17.
Amoebic gill disease (AGD) has been attributed to infection by Neoparamoeba sp. The causal mechanisms for AGD lesion development and the primary pathogenic role of Neoparamoeba sp. require elucidation. Three groups of Atlantic salmon were exposed to viable gill isolated amoebae, to sonicated amoebae, or to sea water containing viable amoebae without direct contact with gill epithelia. Fish were removed 8 days post-exposure and the gills assessed histologically for AGD. AGD occurred only when fish were exposed to viable trophozoites. Consequently, in an accompanying experiment, infection was evaluated histologically at 12, 24 and 48 h post-exposure in three groups of salmon, one group being mechanically injured 12 h prior to exposure. A progressive host response and significant increase (P < 0.001) in the numbers of attached amoebae was apparent over the 48-h duration in undamaged hemibranchs in both treatment groups. There were no significant differences to mucous cell populations. Attachment of Neoparamoeba sp. to damaged gill filaments was significantly reduced (P < 0.05) by 48 h post-exposure. These data further confirm and describe the primary pathogenic role of Neoparamoeba sp. and the early host response in AGD. Preliminary evidence suggests that lesions resulting from physical gill damage are not preferentially colonized by Neoparamoeba sp.  相似文献   

18.
Amoebic gill disease (AGD) of cultured salmonids in Tasmania is caused by the amphizoic parasitic amoeba Neoparamoeba pemaquidensis. The freshwater tolerance of amoebae isolated from the gills of AGD-affected salmon (predominantly N. pemaquidensis) was tested in vitro using a trypan blue exclusion assay. Amoebae exposed to water containing high concentrations of Ca2+ or Mg2+ (200 mg l−1) showed high levels of survival up to 3 h of exposure. Exposure to water containing elevated Na+, choline chloride or water at different pH all had no significant survival of amoebae. Exposure of amoebae to different concentrations of chlorine dioxide, chloramine-T or hydrogen peroxide in artificially hard water demonstrated that chloramine-T and hydrogen peroxide were the most efficacious at killing amoebae in vitro. This work suggests that the hardness of freshwater may be an important factor for the survival of marine amoebae (predominantly N. pemaquidensis) on the gills of AGD-affected salmon and have significant implications with regard to the efficacy of freshwater bathing practices for the control of AGD on farms. Additionally, chloramine-T and hydrogen peroxide appear to be efficacious at killing marine gill amoebae in vitro and may be useful for the control of AGD in farmed Atlantic salmon.  相似文献   

19.
Amoebic gill disease (AGD) of Atlantic salmon is treated commercially by bathing affected fish in freshwater. Recently, the efficacy of freshwater bathing has been questioned, and the aim of this study was to examine the potential for improving bathing efficacy using additives to the freshwater bath. AGD‐affected Atlantic salmon were bathed in 350 L tanks containing oxygenated freshwater to which chlorine dioxide (0–50 mg L?1), chloramine‐T (0–50 mg L?1) or hydrogen peroxide (0–100 μL L?1) was added. Before and following a 3‐h exposure to the freshwater and chemical additive, the gills were removed from a sub‐sample of fish and the number of live amoebae on the gills were counted and smears made for confirmation of the presence of Neoparamoeba pemaquidensis, the causative agent of AGD. Following a further 3‐h exposure, a sub‐sample of fish was bled from the caudal vein and the gills were removed for histological examination. Chlorine dioxide and chloramine‐T at 25–50 and 10–50 mg L?1, respectively, reduced the number of amoebae on the gills by approximately 50% compared with pre‐exposure numbers. The results from hydrogen peroxide treatment were equivocal and the toxicity of hydrogen peroxide was high. The toxicity of chlorine dioxide varied with freshwater hardness and/or suspended solid load, whereas chloramine‐T toxicity was low, with mortalities attributable only to elevated temperatures at the highest concentration tested. In conclusion, chlorine dioxide and chloramine‐T show promise as potential freshwater additives for the improved removal of N. pemaquidensis and possibly, other amoebae from the gills of commercially farmed Atlantic salmon.  相似文献   

20.
Spontaneous mortality of seemingly healthy, farmed Atlantic salmon (Salmo salar L) is an increasing problem in Norwegian aquaculture. In this study, we present a morphological study of the previously undescribed syndrome of arteriosclerosis of the ventral aorta and epicarditis of the adjacent bulbus arteriosus found in farmed Atlantic salmon, with wild‐captured fish as a control group. Both the ventral aorta and epicardium are vital for correct arterial compliance and vascular resistance in the respiratory capillaries of the gills. We discuss the possible implications of ventral aorta arteriosclerosis and epicarditis for blood vascular health and in particular for the increasing frequency of spontaneous gill bleeding in farmed salmon. As both these conditions primarily occur in farmed salmon, we suggest that they should be considered pathological.  相似文献   

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