Healthy gills are important for growth and welfare of salmon. Gills have several vital functions, and multifactorial gill disease is the cause of significant loss of salmon in the seawater phase in Norwegian fish farms. Both infectious and non-infectious factors cause gill disease in sea water. In the project we have developed models for disease development, characterized and studied the importance of different types of organisms that may be involved in disease development.
CHARACTERIZATION AND CULTIVATION OF SELECTED AGENTS
In order to establish good challenge models it is important to be able to cultivate current microorganisms in the laboratory. We have succeeded in establishing a monoclonal culture of the amoeba Paramoeba perurans that causes amoebic gill disease (AGD). Attempts to grow salmon gill pox virus in cell culture have not succeeded. This work is still ongoing. Several intracellular microorganisms associated with gill disease are not possible to grow in the laboratory. One example is the bacteria Branchiomonas cysticola, one of the causes of the disease epitheliocystis in salmon.
A survey has been conducted to characterize agents related to the development of epitheliocystis in gill tissue. The material originated from the Norwegian Veterinary Institute's biobank and was collected in the period 2004-2014. In situ hybridization and laser dissection microscopy were used to isolate bacterial DNA from histological preparations and the 16S rRNA genes sequenced using ?universal? PCR primers. The result confirmed that B. cysticola is the dominating agent of epitheliocystis in Norwegian farmed Atlantic salmon being identified in 83% of the 65 selected cases. Previously undescribed taxa probably representing novel Branchiomonas species were identified in the remaining 17% cases from Atlantic salmon and from Atlantic cod respectively. We are now working on characterization of these novel taxa. Results are so far reported at national and international meetings.
We conducted an electron microscopic study of gills with AGD, which showed that P. perurans cause greater damage to the gill tissue than previously thought. This indicates that the amoeba produces extracellular products that damage the gill tissue (Wiik-Nielsen et al. 2016, doi: 10.1111 / cf.). In collaboration with international partners we have also carried out genetic studies comparing isolates of P. perurans from different localities in several countries. The results from the study showed only minor genetic differences between the different clones.
MODEL TRIALS WITH LIVING FISH
Using the AGD infection model, we conducted model experiments with exposure to jellyfish and hydroids. Jellyfish and hydroids (biofouling) contain sting cells that can insult gills. Jellyfish can bloom during summer and hydroids are released by washing net pens.
Model experiments with Atlantic salmon were conducted to test the following:
1) Effect of exposure with the jellyfish Cyanea capillata on the development of AGD
2) Effect of exposure to the hydroide Ectopleura larynx on the development of AGD
3) Transmission of the bacterium Branchiomonas cysticola from naturally infected fish to näive fish
4) Effect of AGD on hypoxia tolerance in salmon
Summary of results
Preliminary results from Experiments 1, 2 and 4 have been reported at meetings and conferences and are now being published. Briefly, gill irritation was detected by exposure of salmon to jellyfish and hydroids. However, no faster or more severe development of AGD was detected in this pre-exposure trials. However, the results of jellyfish exposure alone showed interesting physiological effects on cardiac function and respiration in salmon. Degree of AGD had no significant effect on the hypoxia tolerance in salmon.
The results of Experiment 3 showed that B. cysticola was transferred horizontally via water and caused epitheliocystis in näive fish. In addition, it was documented transmission of another epitheliocyst-forming bacteria and salmonid gill pox virus in the same experiment (Wiik-Nielsen et al., 2017 doi: 10.1111 / cf.12613).
COMPLEX GILL DISEASES IN THE FIELD
The NVI has also conducted a study of seven cases with complex gill disease in freshwater and seawater farms from different parts of Norway. In addition to gill pox virus, several other gill pathogenic organisms were detected. All sites had high mortality rates. When the pox virus infection coincided with smoltification, mortality became extreme. The results indicate that the pox virus attacks chloride cells and may therefore affect the smoltification. Furthermore, the virus can play a role as a primary pathogen that can pave the way for other gill pathogens (Gjessing et al., 2017, doi: 10.1111 / cfd.12608).
Sound gill health is a key-factor for fish growth and welfare. Gill disease contributes with a significant part of disease-related losses in marine farming of Atlantic salmon in Norway. A better understanding of gill diseases, their etiology, pathogenesis and pathophysiology, is needed to develop more effective disease prophylaxis and treatment. The gills are multifunctional organs, involved in respiration, osmoregulation, acid-base and nitrogen excretion in fish. A number of infectious and non-infectious factors are linked to gill diseases, and the etiology seems to be of multifactorial origin.
The aim of the project is to study the significance and interaction of different factors associated with gill disease, by means of challenge models with live fish . Standardized challenge models will be developed with viral, bacterial, parasitic agents with expected importance for gill disease, as well as with planktonic agents like algae and diatoms, and zooplankton (such as jellyfish and siphonophores). The agent s to be involved in these trials include the amoeba Paramoeba perurans, the bacterium Branchiomonas cysticola, and poxvirus. Cultivation of these agents in vitro for challenge purposes will be emphasized, and they will also be subjected to more detailed g enetic characterization. Potentially harmful phytoplankters will be included, such as Chaetoceros sp. and Alexandrium sp., or jellyfish like Aurelia aurita or Periphylla periphylla.
The effects of challenge will be studied in trials with one agent alone , or more agents in combination. Parameters to be measured include respiratory and cardiovascular responses, and effects on blood chemistry, acid-base balance and electrolyte levels. Histopathological changes will be evaluated by implementing a new gill s coring system based on a totality of cellular and epithelial abnormalities. The detection of relevant gill associated microbial agents will be included.