Introduction
Amoebic gill disease, also known as (AGD) is a parasitic infection of the gills affecting a large number of fish species farmed in marine environments. The infection of these fish is mainly caused by Neoparamoba puritans, a recently described parasitic and free-living amoebae, which is the aetiological agent of amoebic gill disease. The infection by Neoparamoba perurans is mostly characterized by multifocal lesions in the gills, which cause severely compromised gill physiology and functioning. Affected fish suffer from an increased susceptibility to stress (especially during treatment of Neoparamoba perurans), respiratory distress, and finally, mortality. Treatment and control of AGD involve regular monitoring of the gills and prophylactic baths with fresh water.
The Atlantic salmon (Salmo salar) is one of the marine species that is largely affected by AGD (Oldham et al., 2016). The Atlantic salmon naturally occur along the east and west coast of the North Atlantic Ocean, where it exists in anadromous and non-anadromous freshwater resident forms, after which it will go through a process called smoltification and a long migration to the ocean for feeding and growth. Its habitat stretches from the Connecticut River in the south to Ungava Bay in the north. While in the northeast and the north, its habitat ranges from northern Portugal to the rivers that lead to the Barents and white sea area. Atlantic salmon farming traditionally began in the 1960s and became a large industry in countries like Norway, Canada, and Scotland. Nowadays, Atlantic salmon farming also takes place in Australia, New Zealand, and the Faroe Islands. Atlantic salmon are an important part of the industry, and disease prevention is a key concern.
The production of salmon is getting increasingly important as the world population increases to the large extent and will reach about 9.7 billion by 2050, according to the FAO of the UN (Global Salmon Initiative, 2019). The need for protein is expected to grow by 40% worldwide. As the demand for protein increases, so will the strain on the already over-exploited wild fish reserves. Since 60% of the world’s salmon production is farmed, farmed fish is very important in maintaining the natural balance in wild salmon (Global Salmon Initiative, 2019). Salmon is an exceedingly efficient source of fats and protein, as it contains high levels of Omega-3 and lots of protein. Because of this, cultured salmon continues to outperform other protein sectors like the poultry and pork industry (Global Salmon Initiative, 2019).
Nevertheless, growing concerns arise due to the increasing cases of AGD reported in Ireland, France, and Scotland. AGD tends to be the most serious infection health challenge for the marine salmon farming industry as it can cause up to 10% of livestock mortality per week if left untreated (Boison et al., 2019). However, no commercial vaccine is available at the moment, and prevention by treating the fish with prophylactic baths costs the fish farmers an immense amount of time and money (Bustos et al., 2011). This is primarily because some fish batches have to be treated up to 15 times during their marine grow-out cycle. Possible alternatives such as genetic resistance and preventive screening measures are currently being researched in the hope of reducing the effect of AGD on the salmon farming industry.
This review paper aims to give an account of current research being conducted on the prevention and curation of AGD. First of all, the pathology of Neoparamoeba perurans will be elaborated to be able to understand the physiology behind the different treatments. Secondly, different treatments will be discussed, followed by studies that are currently researching possible curation treatments. The final section will continue with recommendations for possible research about AGD treatments.
The Pathology of Neoparamoeba Perurans
Until yet, the aetiological organism, Neoparamoeba perurans, has not been cultured in vitro; hence Koch’s precepts have not been met. The difficulty of producing the amoeba has hampered the development of AGD research, requiring the maintenance of constant experimental infestations to supply infective chemicals. Utilizing malted yeast substrate with seawater top and subculturing each 3–4 days, a colony cell of Neoparamoeba perurans was produced (Valdenegro-Vega et al., 2015). PCR proved the amoebae identity. The diseased Atlantic salmon producing AGD was re-isolated from the fish samples after 70 days in vitro. Histology verified the identification, and PCR and in vitro experiments using universal primers and labels previously established and unique to Neoparamoeba perurans identified the infecting agent. This research demonstrates Koch’s ideas about Neoparamoeba perurans as a source of AGD while also revealing its wild and harmful nature.
Neoparamoeba perurans were isolated from AGD-infected Wild salmon reared in the west of Ireland; the amoeba civilization was built and implemented. A sample population of the colony was analyzed by conventional PCR to indicate the existence of Neoparamoeba perurans (English et al., 2019). Amoebae were extracted from the agar by physically removing them with a bacteriological distributing bar, followed by numerous seawater washes. The amoeba saltwater combination was then kept in a clean beaker. Many measurements of the amoeba saltwater mix were done utilizing a one ml Sedgewick Rafter Measuring Capsule.
The 18S rRNA gene is duplicated several times in the Neoparamoeba Perurans genotype, with an authentic PCR standard curve for suspension cultures and plasmids providing an average of 2880 clones per unit. The new approach was employed to collect seawater specimens from an artificial AGD disease container as well as a range of environmental locations, including those used to grow Atlantic salmon. Perceptible colonies were numerous at places within and adjacent to Atlantic salmon cage cultivation (Marcos-López et al., 2017). Additionally, when performed to gill biopsies from an on-farm gills pathology evaluation, the approach proved the feasibility of non-destructive semi-quantitative characterization of amoebae loads in these fish. The quantitative nature of this innovative technique reveals the influence of sea net farming on the incidence of this salmon disease. It is a step towards confirming the spread of Neoparamoeba Perurans throughout the aquatic organisms and its association with AGD epidemics.
Peak mortality rates for infected Atlantic salmon smolts in Tasmania might approach 10% per week. For example, those results in making (500 g) in Spain might range between 5 and 20% throughout three months, from October to December. Salmonids rise to the water surface, and exhibit increased opercular motions, indicating lethargy and breathing difficulty. Experiment tests, unfortunately, were unable to show that AGD-infected fish had a meaningful increase in breathing rate.
Macroscopic lesions in Atlantic salmon are often multifocal regions of white to grey swollen gill material with concomitant excessive mucus. The dorsal parts of the gill arch have the most of them. Mucoid pneumonia is more common in rainbow trout. Diseased turbots exhibit behavioral changes such as decreased feeding and reversal posture, gills coated in excess mucous, malformed, decaying gill threads, and areas of greyish peripheral discoloration. Salmonid and turbot histopathological lesions are comparable, with proliferation and enlargement of the gill epithelium being the predominant lesion. Neoparamoeba pemaquidensis was found in the normally normal gills of AGD-infected fish two days after exposure (DPE).
During 4 DPE, widespread proliferation and membranous fusion had developed, with up to 15-gill lamellae per fuci involved by 7 DPE. The tissues were spongiotic, with hyperplastic and hypertrophic epithelial cells. Neoparamoeba pemaquidensis was more prevalent, mostly connected with the precancerous endothelium, and was peeled off with sheets of plexuses material in certain cases. A significant increase in mucus cells was also observed. Endothelial hypertrophy and membranous union were widespread at 28 DPE, with several connected Neoparamoeba pemaquidensis. Interlamellar vesicles frequently formed, frequently harboring amoebae and aggressive inflammatory cells in the surrounding bone. Neoparamoeba pemaquidensis is restricted to the gill area, and no histological alterations in internal organs have been seen. The critical metabolic changes linked with AGD are yet unknown. Fish with severe symptoms have high blood salt levels and respiratory alkalosis. These anomalies, nevertheless, are not broad or significant enough to underlie the clinical symptoms.
Symptoms and Diagnosis
Only cultivated varieties appear to be threatened by the illness, with salmon in their first year at sea appearing to be most vulnerable. In the early stages of infection, there are few visible signs; as the disease progresses, indicators of respiratory diseases, such as flared operculate and gasping, can be noted. Fish are frequently found further up in the water layer. Mucoid spots appear on the gills, increasing mortality rates.
It is critical to identify AGD from the presence of N. pemaquidensis with minimal gill disease. The former is the most important diagnostic for aquaculturists, but doctors and, in particular, researchers are interested in both elements of the classification. The usual mucoid spots on the gills are counted to make an on-farm diagnosis. While the prevalence of N. pemaquidensis is substantially connected with this in experimental infections on Atlantic salmon, the connection is unclear in the environment. Grossly apparent lesions are not always present in infected turbot.
There are several diagnostic treatment aims for confirming a medical assessment. An immunofluorescent antibody test can be used to stain wet mounts of fish gills (IFAT). The latter is a little more challenging since amoebae can be hard to distinguish from gill vascular endothelium. In addition, a dot-blot approach has been created. The test looks to be sensitive and selective, making it suitable for mass fish testing. Focusing mucus rinsed out with salt water and placing a little of the loose particle on a glass slide in a moist chamber for 30 minutes allows trophozoites to adhere to the coverslip in a more complicated fashion.
Trophozoites can be viewed immediately under a fluorescent microscope or stained with hematoxylin and eosin (H & E) and DNA labeling after being treated with preservatives. Histological procedures can also be used to evaluate gill specimens. In experimental infections, amoebae can be spotted adhering to the gill epithelial as early as 2 DPE, which is the earliest time for any approach to confirm infection. In 7 DPE, IFAT is present. A unique PCR has been created; it detects the pathogen with 95% accuracy and may be used on freshwater bodies, saltwater, and foulants.
Antiserum antibodies raised against the Tasmanian isolation bind to organisms found on salmon in Ireland, New Zealand, and France. PCR experiments show that microorganisms collected from AGD on rainbow trout in Australia, Ireland, and Washington State, as well as tuna in Spain, are the same. However, because the species from the United States and Spain can live at considerably lower salinities than the Australian N. pemaquidensis, it appears that the test does not discriminate between biovars with diverse biological properties.
Finfish viral infections accumulate and remain in shellfish for lengthy periods. In addition, 13p2 reovirus, comb fish virus, viral pancreatic necrotic isolates, and IHNV have been isolated and characterized as fish illnesses from shellfish. A variety of bacterial pathogens that cause illness in finfish are also found in bivalve tissue. These cephalopods are involved in the membrane fouling mechanism. The intake of various contaminants by cultured species was connected to the onset of net-pen liver disease in caged fish. Biofouling has also been proposed as a possible function for ISA in salmon epidemics.
Prevention
Clean living standards for fish are essential for their health. Keeping clean living standards for fish is the greatest approach to inhibiting the growth of bacteria gill illness (Wynne et al., 2019). Keeping the stream clear of plant matter, providing plenty of space for the fish to move without crowding, ensuring consistent temperature, and testing the water quality regularly to verify that it is balanced are all good practices for maintaining fish healthy and stress-free (Overton et al., 2017). Furthermore, filters must be replaced or examined monthly or as directed by the filter supplier.
Bacterial gill infection must initially be treated by changing the fish’s living environment. If they get overcrowded, they will want extra room, either in a bigger tank or dispersed into multiple aquariums. The hygiene of the aquarium and its water is critical (Rozas-Serri, 2019). To assist the fish heal and recovering from the infection, potassium permanganate and seawater additives might be utilized. The quantity of salt a person use may vary depending on the species they are addressing, but it must be a salt produced particularly for fish freshwater and only in the appropriate amount. Secondary bacterial infections may be treated with antibiotics.
Currently, the most successful treatment is to place the sick fish in a water bath for two to three hours. To do this, the water cages are towed into freshwater, or the fish are pumped from the sea cages into a tarpaulin full of freshwater (Powell et al., 2015). The death rate has been reduced by introducing levamisole to the water until the saturation level exceeds 10ppm (Overton et al., 2018a). Access to stream freshwater limits the profitability of salmon aquaculture due to the complexity and price of treatment (Wright et al., 2018). Chloramine and chlorinated dioxide have been employed as well. Other possible in-feed therapies, such as immunological caring diets, antimuscarinic chemicals like L-cysteine esterification, and the parasiticide bithionol, have been investigated with some effectiveness but have not yet been commercialized.
Hydrogen peroxide was proven to be effective as a therapy for AGD in all dosages and temperature combinations examined. Following therapy, the gills partially healed, and the bacteria’s progression was slowed (Martinsen et al., 2018). However, following treatment, all classes of fish got the sickness again, indicating that the fish were not healed (Overton et al., 2018b). Because the salmon is already compromised by the sickness, both freshwater and hydrogen peroxide therapy may result in death, and the therapy itself might cause extra stress to the fish (Vera and Migaud, 2016). AGD seems to resurface in treated fish under both natural and regulated experimental situations, indicating that neither therapy is completely successful. Moreover, it is critical for both financial and fish well-being reasons that the approach utilized is both successful and friendly to the fish.
The temperate surface water is most effective for submerging Atlantic salmon in AGD, and the bulk of Norway’s freshwater habitats are soft. Because of their low acidity and neutralizing capacity, such streams may affect the pH and heavy metal toxicity in the water supply (Lillehammer et al., 2019). Additionally, soluble organic material can be advantageous in therapy, albeit metal ion sequestration can be reversed when the pH of the water declines to owe to high fish populations and atmospheric carbon abundances.
Experimental treatment for AGD and ocean lice control, such as the use of reactive antibacterials such as hydrogen peroxide, may hold promise, albeit the correlations with organic loads and soluble plant molecules in seawater are unclear. Furthermore, the usage of oxidative antiseptics in freshwater will be determined by the chemistry of the water and reactions with treatment agents, fish, and natural moisture content (Robledo et al., 2020). Large organics of Atlantic salmon in ocean farming provide logistical challenges. Although the use of decent boats has promise, preserving the quality of the water throughout treatments is critical for both AGD and sea lice therapies to maximize fish wellbeing and therapy success.
Avaricious gill and cutaneous blood miscues lay fertile eggs, which are then discharged into the water, producing visible knotted mounds at times. These eggs have a tough shell and long tendrils that finish in hooks, allowing them to connect straight to netting or fouling on the netting (Nowak et al., 2014). Diseased fish are routinely bathed in various dangerous chemicals in fish farms. Unfortunately, this removes the parasite’s pathogenic phases from the fish, abandoning unfertilized eggs connected to the netting undisturbed. These eggs will develop and infect the fish in the cage once more.
Thus, coordinating fish baths with net maintenance is critical for extending the times when the fish are not affected. Another preventative technique is to employ netting material, which considerably decreases the capacity of parasite eggs to colonize it efficiently. In Hawaiian offshore mariculture, this has been investigated for Neobenedenia. Small spaces generated by braided rope strands allow for egg adhesion, whereas surface texture netting substances offer quicker extraction of eggs and filth with dynamic cleaning solutions such as power washing. An antifouling component put on the nets or constructed of an antifouling element, such as copper, dramatically decreased egg adhesion.
In reality, the deceased versus surviving state after an infected test is the attribute most commonly employed to measure personal susceptibility or vulnerability to an illness, particularly for viral or bacterial infections. Fish that die throughout a challenge are categorized as vulnerable, while those that survive are classified as resistant. Resistance is thus assessed as a binary qualitative feature for this evaluation. It is frequently supplemented by the time measurement, which seeks to better distinguish the possible variance incapacity to regulate illness dynamics and the formation of harmful consequences within dead fish (Treasurer and Turnbull, 2018). It must be mentioned that the true circumstances of live fish are often unclear.
Conclusion
To conclude, amoebic gill disease (AGD) is a parasitic disease of the membranes that infects several marine-farmed marine species. Neoparamoba perurans, a recently characterized infectious and unrestricted amoeba that causes amoebic gill sickness, is the most characteristic of the disease in these fish. Neoparamoba perurans infection is characterized mostly by widespread lesions in the gills, resulting in significantly reduced gill physiology and functionality. One of the fish organisms most impacted by AGD is the Atlantic salmon. The Atlantic salmon is found naturally throughout the east and west coasts of the North Atlantic Ocean, where it occurs in anadromous and non-anadromous freshwater permanent stages before migrating to the open sea for eating and growth in a process known as smoltification.
Nonetheless, increased concerns have arisen as a result of an increase in AGD cases recorded in Ireland, France, and Scotland. AGD is the most significant infectious health concern for the saltwater salmon farming industry, causing up to 10% of animal death each week if neglected. Unfortunately, no commercial vaccination is now accessible, and treatment using prophylactic baths costs fish producers a significant amount of time and money. This is mostly because certain fish groups must be medicated up to 15 times throughout their marine development period.
The difficulty of producing the amoeba has hampered the development of AGD research, necessitating the maintenance of a constant laboratory-based infestation to supply infective chemicals. Applying malt yeast agar with seawater overlay and subculturing every 3–4 days, a clonal culture of Neoparamoeba perurans was produced. PCR confirmed the identification of the amoebae. After 70 days in vitro, the sick Atlantic salmon generating AGD was re-isolated from the fish samples.
Neoparamoeba perurans were produced from AGD-infected Atlantic salmon farming in the west of Ireland; the amoeba civilization was constructed and deployed. A colony sample population was examined using conventional PCR to determine the presence of Neoparamoeba perurans. Using a biological dispersion bar, amoebae were forcibly removed from the agar, accompanied by several seawater applications. In the Neoparamoeba Perurans genotype, the 18S rRNA genome is multiplied multiple times, with a real PCR calibration solution for cell suspensions and plasmid DNA yielding a value of 2880 transcripts per cellular.
Maximum deaths for diseased Atlantic salmon smolts in Tasmania might be as high as 10% each week. For instance, those in manufacturing (500 g) in Spain might fluctuate between 5 and 20% over three months, from October to December. Macroscopic tumors in Atlantic salmon are often multifocal regions of pale to beige dilated gill epithelium with contemporaneous copious mucus. The majority of them are found in the dorsal sections of the gill arch. Rainbow trout are more prone to mucoid pneumonia.
Only cultured kinds appear to be at risk, with salmon in their first year at sea proving to be the most vulnerable. In the early stages, there are few visible indications of the sickness; however, as the disease advances, markers of respiratory disorders, such as flared operculate and gasping, can be observed. It is crucial to distinguish AGD from N. pemaquidensis with minor or no gill disease. The former is the more appropriate diagnosis for aquaculturists, but both aspects of the categorization are of importance to clinicians and, in particular, researchers. To make an on-farm diagnosis, the normal mucoid patches on the gills are counted.
Maintaining clean living conditions for fish is the most effective way to prevent the spread of bacteria gill disease. Keeping the channel clear of plant matter, allowing enough space for the fish to move without overcrowding, keeping a constant temperature, and monitoring the water quality regularly to ensure that it is balanced are all essential processes for fishkeeping alive and stress-free. Potassium permanganate and saltwater additives might be used to help the fish mend and recover from the illness. The amount of salt used may vary depending on the species, but it must be a salt created specifically for freshwater fish and only in the proper amount.
Today, the most effective therapy is to immerse the ill fish in a steam bath for two to three hours. To do this, the sea cages are dragged into freshwater, or the salmon are pushed from the sea cages into a tarp full of surface water. Hydrogen peroxide was shown to be successful as an AGD treatment in all dose and temperature configurations tested. The gills partially recovered after treatment, and the bacteria’s development was delayed. However, after therapy, all types of fish became ill again, showing that the fish had not been cured. Because the fish is already weakened by the illness, both freshwater and hydrochloric acid treatment may end in mortality, and the treatment itself may create more stress.
Therapeutic approaches for AGD and ocean lice control, like the use of oxidative antiseptics like hydrogen peroxide, may hold promise, albeit the correlations with natural loads and dispersed plant molecules in seawater are unclear. Moreover, the biochemistry of the fluid and reactivity with treatment agents, fish, and natural moisture content will influence the use of oxidative antiseptics in freshwater.
Pairing salmon pools with netting maintenance is critical for extending the time the salmon are not damaged. Another protective strategy is to employ netting mesh, which decreases disease eggs’ capacity to populate successfully. Small holes created by braided rope strands allow for egg attachment, whilst surface texture netting materials enable faster egg and dirt evacuation using active cleaning agents such as power washing. Salmon that succumb throughout a test are branded as responsive, while those that survive are classed as robust. For this evaluation, resistance is thus appraised as a binary qualitative attribute. It is typically augmented by time measurement, which tries to better identify the potential variance in the inability to manage sickness dynamics and the production of adverse consequences inside dead fish. It should be mentioned that the true circumstances of live fish are often unknown.
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