A cohort study of gill infections, gill pathology and gill‐related mortality in sea‐farmed Atlantic salmon (Salmo salar L.): A descriptive analysis

Abstract Gill disease is an important cause of economic losses, fish mortality and reduced animal welfare in salmonid farming. We performed a prospective cohort study, following groups of Atlantic salmon in Western Norway with repeated sampling and data collection from the hatchery phase and throughout the 1st year at sea. The objective was to determine if variation in pathogen prevalence and load, and zoo‐ and phytoplankton levels had an impact on gill health. Further to describe the temporal development of pathogen prevalence and load, and gill pathology, and how these relate to each other. Neoparamoeba perurans appeared to be the most important cause of gill pathology. No consistent covariation and no or weak associations between the extent of gill pathology and prevalence and load of SGPV, Ca. B. cysticola and D. lepeophtherii were observed. At sea, D. lepeophtherii and Ca. B. cysticola persistently infected all fish groups. Fish groups negative for SGPV at sea transfer were infected at sea and fish groups tested negative before again testing positive. This is suggestive of horizontal transmission of infection at sea and may indicate that previous SGPV infection does not protect against reinfection. Coinfections with three or more putative gill pathogens were found in all fish groups and appear to be the norm in sea‐farmed Atlantic salmon in Western Norway.

However, the exact cause(s) and pathogenesis of a considerable proportion of gill disease cases are unknown (Boerlage et al., 2020).
The understanding of the roles of Ca. Branchiomonas cysticola and D. lepeophtherii in the development of gill disease is limited by the lack of established challenge models and the fact that both agents are nearly ubiquitous in sea-farmed salmon in Northern Europe (Downes et al., 2018;Gunnarsson et al., 2017;Mitchell et al., 2013;Steinum et al., 2010). Whilst these agents are detected in salmon without gill disease, higher pathogen loads have been reported in diseased gills and the microorganisms have been detected in association with gill lesions using in situ hybridization (Gjessing et al., 2021;Mitchell et al., 2013;Weli et al., 2017).
Monitoring of zoo-and phytoplankton levels is not commonly performed at Norwegian sea farms and whether zoo-and phytoplankton levels not associated with acute severe mortality can impact gill and fish health has not been explored.
Farmed Atlantic salmon will frequently be exposed to a range of potential insults and environmental factors during a production cycle at sea. Each might have an impact separately, but they can also coincide in time and interact to determine whether fish will develop clinical disease or mortality. Cohort studies are especially useful for evaluating the relationship between exposures and the development of disease, and to observe change over time. Further, the sequence of events can be established and it may be possible to identify and link events (like gill disease) to a particular exposure (Caruana, Roman, Hernández-Sánchez, & Solli, 2015). However, relatively few longitudinal studies focusing on gill health including multiple sites and fish groups and with a parallel sampling of fish for histopathology and RT-qPCR have been performed.
In order to gain more knowledge about factors affecting the gill health of Atlantic salmon in Norwegian aquaculture, we performed a prospective cohort study, following 16 fish groups from 8 sea farms with repeated sampling and data collection from the hatchery phase and throughout the 1st year at sea. The objective of this study was to determine if variation in pathogen prevalence and load of N. perurans, Ca. B. cysticola, D. lepeophtherii and SGPV, and water concentration of jellyfish and phytoplankton had an impact on gill health as measured by gill-related mortality, gross gill scores and extent of tissue damage detected by histopathology. The secondary aim was to describe the temporal development of pathogen prevalence and load of N. perurans, Ca. B. cysticola, D. lepeophtherii and SGPV, observation of pathogens, and different types of gill pathology, and how these relate to each other. In this article, we report the descriptive analysis of these data and the associations between gross gill scores, extent of gill histopathology and pathogen load in individual fish.

| Study population
The main study unit was the 'fish group', defined as fish from the same hatchery transferred to sea at the same time and to the same cage. Parallel gross scores, histopathology and RT-qPCR results from individual fish additionally allowed for examination of the correlation between tissue lesions, pathogen prevalence and load using 'fish' as the study unit.
Four freshwater sites and eight seawater sites were included in the study. Fish groups were split into two pens at the same site at sea transfer, resulting in eight separate fish groups (A-H) during the freshwater phase and 16 fish groups (A1-H2) during the seawater phase of production ( Figure 1). Freshwater sites were selected to include two sites using flow-through systems (FT), two using recirculating aquaculture systems (RAS), two sites with a history of gill disease (sites 3 and 4) and two sites without known gill issues (sites 1 and 2). From each freshwater site, one fish group sea transferred in autumn 2018 (S0) and one fish group sea transferred in spring 2019 (S1) were included.
A winter signal was given at site 1 (12 h light:12 h dark for 5 weeks, S0 fish only) and 3 (12 h light:12 h dark for 6 weeks), and salinity was increased prior to sea transfer to facilitate smoltification at sites 2-4. Sea sites were selected to include four sites with a history of problems with gill disease and gill-related mortality (sites A, C, D and G), whereas the remaining four other sites only had mild or no recorded historic gill-related mortality. Sea sites were in Western Norway and fish groups at each site were sea transferred at ap-

| Environmental data
Sea temperatures were recorded daily for each sea site, whereas salinity was only repeatedly recorded for sea sites B and C varying from >1 month to 1 week between recorded measurements. Measurements of salinity at the remaining sites were not available or very limited.

| Mortality data
Mortality data from all sites were retrieved through the management database Mercatus Farmer (ScaleAQ, Norway). The total number of fish dead per day (total mortality), the number of dead fish in each mortality category (cause-specific mortality) per day and the number of stocked fish per day were provided for each site and pen. Causespecific mortality was generated by site staff daily assigning a likely cause of death based on gross examination of dead fish and knowledge of infectious disease and management events occurring at the site. Each fish was only assigned one mortality cause and if the classification of mortality to one category was not possible, mortalities were classified as 'unknown' or 'other'. Fish health personnel gave recommendations about classification and were further involved if there were any significant mortality events. Following visits by fish health personnel and/or results from laboratory analysis, the mortality categories could have been retrospectively adjusted. Results of RT-qPCR analysis performed as part of this study were made continually available to sites and may have been used when categorizing mortalities.

| Sampling of fish
Each fish group was followed prospectively with regular sampling in the freshwater (FW) and seawater (SW) phases, whereas regular gross gill scoring was performed in the sea phase only. In the freshwater phase, 20 to 30 fish per fish group were sampled 0 to 3 times for histopathology (n = 350) and RT-qPCR (n = 378) analysis.
For a sample overview, see Table S1. Fish groups B and C were not sampled during the FW phase. The time from sea transfer until first sampling at sea ranged from 20 to 84 days across fish groups and the time between each sampling ranged from 20 to 122 days. About 10 to 30 fish were sampled per fish group and at 6 to 10 time points at sea. Fish groups A2, B1, B2 and F1 were mixed with fish from other cages during summer and autumn 2019, whereas fish groups G1, G2, E1, E2 and F2 were split into smaller groups during spring, summer or autumn of 2020. During each splitting or mixing event, the pen with the most fish from the original fish group was designated the project pen and fish group and followed moving forward.
Most mixing events occurred late in production, with only one sampling performed after mixing for three fish groups (A2, B1 and B2), whereas six sample sets were collected after mixing of fish group F1. After the mixing, 49% (B2), 64% (A2), 85% (B1) and 95% (F1) of the original fish group remained in the designated project pen.
At each sampling point, the aim was to sample up to 15 fish with clinical signs of disease and 15 presumed healthy fish per pen/ tank, but no fish sampled during FW and very few fish sampled in SW showed clinical signs of disease (n = 44) and most were presumed healthy (n = 2075) or of unknown health status (n = 1814). In the freshwater phase, fish were sampled using a dip net. At the sea sites, fish in the net pens were crowded using feeding and a purse seine, and fish were selected from the seine with a dip net, as described in the standard operating procedure of the fish farms. Prior to tissue collection, fish were killed by placing them in anaesthetic bath until dead. Within 5 minutes after euthanasia, the second left gill arch was sampled for histology, whereas tissue samples from the third left gill arch were placed in RNAlater (Sigma-Aldrich) for RT-qPCR analysis. Gross gill scoring was performed on anaesthetized fish prior to tissue sampling as far as practically possible (n = 3593).

| Gross gill scoring
A gross gill score system based on the total area of abnormal tissue in the gill was adapted from a system developed by Fish Vet Group UK (personal communication Angela Ashby). Each left gill arch (both F I G U R E 1 Overview of freshwater sites (n = 4), sea sites (n = 8) and fish groups (n = 16). Stocking period (S0 = autumn stock/S1 = spring stock) and water treatment (flow through vs. RAS = circular arrow) is indicated surfaces) was scored separately on a scale from 0 to 5 and then, a mean gill score for all arches was calculated. For each scoring session and fish group, the median of the mean gill score and the proportion of fish with a mean gill score higher than 1 were calculated. Lesions counting towards the score and score categories are outlined in Table 1. Gross gill scoring of 20 fish per fish group was planned to be performed weekly during the sea phase, but between 6 and 50 fish were scored per time point and fish group, with scores available from 33 to 51 weeks across all fish groups (n = 15,553). Scorers included site staff, Mowi project participants, attending fish health personnel and veterinarians and varied from site to site and over time. The majority of scorers received a training session and example images of the different lesions to be scored and categories were provided to each site.

| Histopathology
Gills were fixed in 10% buffered formalin (4% formaldehyde, 0.08 M sodium phosphate, pH 7.0), processed routinely, and sectioned, stained with haematoxylin-eosin (HE) and scanned for histopathologic examination as previously described in Østevik et al. (2021). The study pathologists were 'blinded' regarding results of RT-qPCR-analysis, gross score or water analysis if available. Two different pathologists (HH and MA) examined samples from the freshwater phase, whereas a third pathologist (LØ) examined all samples from the sea phase.
A slightly modified two-step assessment protocol developed by the authors was used (Østevik et al., 2021). Briefly, first, the number of lamellae available for evaluation in each sample was estimated, and then, all affected lamella with hyperplasia or hyperplasia and inflammation, necrosis and vascular lesions (thrombi and aneurysms) were counted (Figure 2). For details and definitions of the type of lesions recorded, see File S1. These counts were used to calculate the estimated percent of gill tissue affected for each type of lesion.
A total histology count was calculated by summarizing the number of lamellae with the lesions listed above and subtracting the number of lamellae with more than one lesion. The total percent affected • Foreign material present between lamella associated with tissue reaction Any pathogens or microorganisms observed in or associated with the gill tissue were recorded as present or absent. For further work with the histopathology data on fish group level the median total percent affected tissue, median percent hyperplasia, median percent vascular lesions and median percent tissue with concurrent hyperplasia and vascular lesions per sampling point were used. In addition, the proportion of fish with the above lesions and pathogens as well as the proportion of fish with more than 5% total affected gill tissue was calculated for each fish group and sampling point and for the fish group overall.

| RT-qPCR
Gill samples from the fresh water and sea water were examined with RT-qPCR for Candidatus Branchiomonas cysticola and salmon gill poxvirus, whereas samples from sea water, in addition, were analysed for Desmozoon lepeophtherii and Neoparamoeba perurans.
Nucleic acid extraction and RT-qPCR-analysis were performed as reported previously (Østevik et al., 2022).  TA B L E 1 Gross score system. The lesions counting towards the score, score categories and the extent of gill tissue affected for each score category are outlined

| Non-medicinal delousing
Information on the treatment method, start date and number of sea lice treatments per fish group was provided by the farming company.

| Plankton sampling and analysis
Sampling of sea water for classification and quantification of gelatinous zooplankton (jellyfish) and phytoplankton was conducted throughout the sea phase. In addition, farm staff was asked to record observations of jellyfish blooms occurring during the study period. A total of 29 to 56 water samples per site were examined for the presence of jellyfish and phytoplankton (n = 323 and n = 322).
Plankton was collected using 250μm mesh nets, with 25 cm and 50 cm diameter ring for phyto-and zooplankton, respectively. Two  States). Line plots were produced to provide a visual presentation of histopathology results, RT-qPCR results, gross scores, mortality data, water temperature, zoo-and phytoplankton levels and management procedures per fish group over time at sea. Trends for the development of gill infections, gill-related mortality, gross scores and gill histopathology over time and by season were described based on these graphs. Possible covariation and associations between the three main gill outcomes; gill-related mortality; gross scores and histopathology results, and between exposures pathogen prevalence and density and zoo-and phytoplankton levels were also assessed at fish group level based on these graphs.

| Gill health in the freshwater phase
Histopathological lesions were absent or minimal to mild suggestive of overall good gill health in all the sampled fish groups (n = 6). One fish group (D) experienced SGPVD-related mortality (3.36% accumulated mortality), but the mortality had ceased when project samples were collected. SGPV was detected with RT-qPCR in four fish groups (C, D, G and H), from two freshwater facilities (sites 3 and 4).
The prevalence of infection ranged from 0 to 53% across fish groups and time points. Ca. B. cysticola was only found in fish group E. No gill-related mortality, nor severe histopathological lesions or high gross gill scores were detected during the sea phase for fish groups D1 and D2, the fish groups that experienced gill-related mortality due to SGPVD in the freshwater phase.

| Environmental data
Sea temperatures during the project period ranged from 4.2 to 17.7°C across all sites and time points and followed a clear seasonal pattern ( Figure 3).

| Gill-related mortality
Presumed gill-related mortality was observed in 10 of 16 fish groups (Figures 3a and b), whereas no gill-related mortality was recorded at sites C, D and F. The mortality assigned as gill-related was generally low with the accumulated gill-related mortality ranging from 0.04 to 1.69% (Table S2). Gill-related mortality was observed both in the spring, summer, winter and autumn months. The highest gill-related mortality occurred at sites G and H. Mortality at site G coincided and followed a peak in severity and extent of histopathology lesions and a high prevalence and load of N. perurans. The gill-related mortality at site H occurred late in the production cycle when sampling and gross scoring were completed, thus the extent of gill pathology and prevalence and load of potential pathogens at the time were unknown.

| Gross gill pathology
Median gross gill scores across the project period were mostly below 1 ( Table 2), but in five autumn-transferred fish groups (A1-2, B1-2 and C1), more than 50% of the scored fish had a mean gill score higher than 1 towards the end of the production cycle (Figures 3c and d). In groups A1, B1 and B2, the increase in gross pathology coincided with low levels of gill-related mortality. Fish group C1 developed bacterial branchitis and likely gross gill lesions as part of systemic bacterial infection, but mortality associated with this condition was recorded as infectious disease-related mortality. No increase in gross gill scores was evident during the time period when sea site G experienced gillrelated mortality, the increased extent of gill histopathology and a high prevalence and load of N. perurans. Overall, gross gill scores did not appear to show a consistent seasonal variation or consistent covariation with water temperature, or prevalence of the putative gill pathogens detected by RT-qPCR analysis.

F I G U R E 3
Sea temperature, daily gill-related mortality (%), percentage of fish with gross gill score >1 and percentage of fish with >5% of gill tissue affected (histopathology) over time at sea per fish group. Daily gill-related mortality (%) for (a) autumn-transferred fish and (b) spring-transferred fish, percentage of fish with gross gill score >1 for (c) autumn-transferred fish and (d) spring-transferred fish, percentage of fish with >5% of gill tissue affected (histopathology) for (e) autumn-transferred fish and (f) spring-transferred fish. The mean daily sea temperature across all study sites is shown in each figure Sea temperature (Celsius) 01apr2019 01oct2019 01apr2020 01oct2020 01jul2019 01jan2020 01jul2020

| Gill histopathology
The number and prevalence of fish with different lesions and pathogens observed and median, minimum and maximum lesion counts and percent across all samples are shown in Tables 3 and   4. The prevalence of lesions and pathogens per fish group is available in Table S2. There was a tendency towards a seasonal variation with more gill lesions observed in autumn and winter. The most severe gill lesions were also detected during or following periods of high sea temperatures in autumn and winter months (Figures 3e and f).
The histopathological lesions observed in the fish groups were generally mild, with the exceptions of fish groups G1 and G2 and to a lesser extent C1, A and B. The median total percent affected gill tissue was generally <2% across all time points and fish groups but ranged from 0 to 52% amongst individual fish. The exception to this was fish groups G1 and G2, where an increase in the median percent affected tissue to higher than 8% was observed during late autumn and winter of 2019. The pattern was similar for the proportion of fish with more than 5% of gill tissue affected. The first autumn and winter at sea more than 60% of fish had lesions affecting at least 5% of gill tissue at site G. In contrast, less than 10% of the sampled fish had this extent of gill lesions at any time in most of the remaining groups (Figures 3e and f).
The median percent lamellar epithelial hyperplasia and/or hyperplasia and inflammation generally showed a similar pattern as percent total tissue affected across fish groups and time points, though more vascular lesions than hyperplastic lesions were observed at some sites and time points. The highest median percent hyperplasia was found in groups G1 and G2, coinciding with the highest median total gill tissue affected. Median percent vascular lesions were less than 1% for all fish groups and time points, and the highest median percent vascular lesion was found in group G1 concurrently with the highest median percent tissue affected. In general, the median percent vascular lesions appeared to increase with increasing time at sea for autumn-transferred fish, but this pattern was not evident for spring-transferred fish. The extent of gill tissue with vascular and hyperplastic lesions in individual fish ranged from 0 to 30% and 0 to 52%, respectively. Overlap of hyperplastic and/or inflammatory lesions and vascular lesions were found in 34% of fish overall but did not constitute a substantial part of the histopathology observed in most fish groups.
Necrosis of lamellae was rarely observed (0.44% of gills sampled) and did not substantially contribute to the total percent affected gill tissue. Necrotic lamella was most often associated with bacterial infection and/or foreign material trapped between filaments No consistent increase in the proportion of fish with deformed filaments was observed over the time at sea. Chronic inflammation of the filaments was observed in more than 40% of fish from fish groups A1-A2 and B1-B2 during the second autumn at sea coinciding with an increase in hyperplastic and vascular lesions. An increase in the prevalence of lamellar oedema was seen in fish groups C1, F1 and F2 at different time points. The increase coincided and was associated with lesions of bacterial branchitis for fish group C1, but no specific disease diagnosis, changes in pathogen density or management operations were associated with the increase in fish groups F1 and F2.

| Gill pathogens
The prevalence of microorganisms observed in tissue sections across fish groups and per fish group is shown in Table 4 and Table S2. The prevalence, median reverse Ct-values and number of fish with mild, moderate and high pathogen load detected by RT-qPCR across all fish groups are available in Table 5. The median reverse Ct-values and prevalence per fish group over time are shown in Figure 5 and However, all fish groups tested positive for SGPV at least once at sea even if they were negative prior to sea transfer. Further, in several fish groups, SPGV appeared to disappear in late winter and early spring before being detected again during the following summer. The presence and load of the parasite and the diagnosis of AGD by histopathology showed a seasonal distribution, being detected during late summer, autumn and winter and disappearing in spring and early summer (Figures 5g-h and 6). A histopathologic diagnosis of AGD or detection of the parasite by RT-qPCR was not necessarily coinciding with severe and extensive lamellar epithelial hyperplasia ( Figure 6), or gill-related mortality or increased gross gill scores.
However, markedly higher median reverse Ct-values in fish groups G1 and G2 (Figures 5g and h) coincided with a high prevalence of N.
perurans infection and AGD diagnoses, the most severe histopathological lesions and gill-related mortality.
Excluding the fish with pasteurellosis from site C and intraepithelial intracytoplasmic bacteria (epitheliocysts), bacteria were sporadically TA B L E 3 Overview of histopathology lesions recorded as counts and percent for all fish groups across the sea phase of the study (n = 3897)   Table 4). No clear seasonal distribution was evident, but the most fish with Trichodina spp. and Ichthyobodo spp.
were found at site G when the most severe gill histopathology was detected.  The presence of foreign material was only recorded when the material was associated with a tissue reaction as for instance inflammation or hyperplasia.

| Non-medicinal delousing
The type and number of sea lice treatments (excluding in-feed treatment) per fish group are summarized in Table S2. The number of delousing operations during the sea phase ranged from 0 to 11 between fish groups, with a median of five treatments per group.
Freshwater and hydrogen peroxide bath treatments that may impact the prevalence and median Ct-values of N. perurans, the prevalence of AGD and development of hyperplastic gill lesions, were performed in 13 fish groups (Table S2). A reduction in the proportion of N. perurans and AGD positive fish was seen at the sampling points immediately after one or more freshwater or hydrogen peroxide  AGD: Prevalence (%) per fish-group 01apr2019 01oct2019 01apr2020 01oct2020 01jul2019 01jan2020 01jul2020 01jul2019 01jan2020 01jul2020 01jul2019 01jan2020 01jul2020 Fish (%) with >5% of hyperplasia 01apr2019 01oct2019 01apr2020 01oct2020 01jul2019 01jan2020 01jul2020 01jul2019 01jan2020 01jul2020 bath treatments in six fish groups, whereas an increase was seen in five groups (Figure 6).

| Jellyfish and phytoplankton
The levels of plankton were generally low with the highest levels of phytoplankton detected during spring and summer at sites C and D (data not shown). The number of microscopic jellyfish was < 50 organisms/m 3 with the highest number during the spring months.
Based on the submitted samples, there were no apparent phytoplankton or jellyfish blooms during the study period. Furthermore, observation of jellyfish blooms was not reported from any of the sea sites. No increase in gross gill scores, gill histopathology, gill-related mortality or total mortality was evident following peaks in total phytoplankton levels to 732,164 and 535,779 cells/L detected at sites C and D, respectively. Additionally, there was no covariation between phytoplankton levels and any of our gill indicators.

| DISCUSS ION
Neoparamoeba perurans appeared to be the most important cause of gill pathology and specifically lamellar epithelial hyperplasia in this study, whereas no consistent covariation and no or weak associations between extent of hyperplasia and prevalence and load of SGPV, Ca.
B. cysticola and D. lepeophtherii were observed. There were no or weak associations between vascular lesions and overlap of vascular

| The impact of gill pathogens on gill health
Neoparamoeba perurans is an important cause of gill disease and gill pathology in salmonid aquaculture, and this has been firmly established since amoebic gill disease was first described in Tasmania (Munday, 1986;Young et al., 2007). Thus, the finding that high loads of N. perurans were associated and coincided with hyperplastic gill lesions and gill-related mortality was unsurprising. Our findings are also in agreement with two longitudinal studies from marine farms in Ireland and Scotland in which increasing N. perurans loads was associated with increasing gill histology scores (Downes et al., 2018;Herrero-Fernández, 2019). N. perurans load and prevalence of amoeba and AGD pathology in tissue sections at group level were highly correlated, indicating an increasing prevalence of amoeba and AGD lesions with increasing pathogen load. However, as tissue sampling was standardized, different gill arches were collected for histopathology and RT-qPCR, and AGD was only diagnosed when amoeba was observed in the tissue section, the true prevalence of N.
perurans and AGD in our fish groups was likely higher than reported here, and an even stronger association between RT-qPCR and histology would be expected if the same arch was sampled for these analyses (Adams, Ellard, & Nowak, 2004;Fringuelli, Gordon, Rodger, Welsh, & Graham, 2012).
As in the current study, increasing Ca. B. cysticola loads was not  We did not find a moderate to strong association or consistent covariation between the extent of gill pathology, gross gill score or gill-related mortality and D. lepeophtherii loads in the current study.
This is in contrast with previous studies reporting that higher D.

| The impact of season and sea temperature on gill pathogens
Neoparamoeba perurans infection in our fish groups showed a seasonal variation with the highest prevalence and pathogen load in autumn and winter, and clearing of infection in spring. This is consistent with the pattern observed in Norway since AGD first became endemic and is likely related to the seasonal variation in sea temperatures (Clark & Nowak, 1999;Mo et al., 2015;Sommerset et al., 2021). Our results also further demonstrate that N. perurans infection and AGD can be self-limiting and resolve without treatment when sea temperatures fall (Clark & Nowak, 1999). Sites C and H with the lowest recorded salinities had the lowest overall prevalence of N. perurans infection and AGD. Unfortunately, information about salinity at most sites and variation in salinity at site H over time was not available, but the low salinity likely explains the markedly lower prevalence of AGD at sites C and H (Clark & Nowak, 1999;Mo et al., 2015;Oldham, Rodger, & Nowak, 2016).
In agreement with previous studies, there was a very high prevalence of D. lepeophtherii and Ca. B. cysticola infection in Southern and Western Norway, and infection was established after sea transfer and persisted amongst all fish groups (Gunnarsson et al., 2017;S. Nylund et al., 2011;Steinum et al., 2015;Steinum et al., 2010;Sveen, Overland, Karlsbakk, & Nylund, 2012

| The impact of the site on gill infections, gill pathology and gill-related mortality
The patterns of gill infections, gill pathology and gill-related mortality within each farm site were markedly more similar than between sites indicating that factors relating to the site are important for these outcomes. This finding is perhaps not surprising as local environmental conditions and infection pressure likely have a considerable impact on gill health and gill infections. Fish groups at the same farm experience a similar environment and are generally exposed to the same type and number of management operations like net cleaning and delousing treatments. These fish also originate from the same stock and hatchery and had experienced similar environmental conditions and exposure to infectious agents before sea transfer. Further, staff performing gross gill scoring and cause-specific mortality classification may also contribute to differences between sites because the local staff at each farm performed these tasks.

| Lacking impact of plankton levels on gill health
In the current study, there was no discernible impact of zoo-and phytoplankton on any of the gill outcomes or the total mortality. However, mostly low concentrations of plankton and jellyfish were detected, and Pseudo-nitzschia spp. and other diatoms were the dominating type of phytoplankton observed. Pseudo-nitzschia species can produce toxins (Amnesic Shellfish Toxins (AST), domoic acid (DA)) that can accumulate in the marine food chain and in mussels.
The toxins can cause serious disease in humans consuming shellfish but are not reported to be associated with fish mortality or disease in Norwegian waters (Karlson et al., 2021). Whilst brief and transient phytoplankton and zooplankton blooms might have been missed because of our sampling regime, the lack of association between plankton levels and gill pathology and mortality in the current study is likely related to the lack of a substantial plankton bloom at any of the sites during the study period.

| Gill-related mortality and gill gross score as indicators of gill health
Gill-related mortality, gross gill scores and extent of tissue damage detected by histopathology were used as indictors of gill health status in this study. We found overall weak correlations between gross scores and the extent of microscopic gill lesions, and low correlations and little covariation between pathogen load and prevalence and gross scores. Our results contrast with several studies reporting good agreement between gross scores and histopathological scores, and gross scores and pathogen load in fish with moderate to severe AGD and N. perurans infections (Adams et al., 2004;Bridle, Crosbie, Cadoret, & Nowak, 2010;Clark & Nowak, 1999). However, Krol et al. (2020) examined the gill arch with the most gross lesions and found no difference in gill histopathology or gene expression in fish receiving different proliferative gill disease (PGD) gross scores, and questioned the usefulness of the PGD gross scoring system for diagnosis and monitoring of gill disease. Mild, focal lesions and diffuse inflammation or hyperplasia can be difficult or impossible to detect grossly (Clark & Nowak, 1999). Thus, the lack of severe gill pathology and high pathogen load in most of the sampled fish likely contributed to poorer correlation between gross and histopathological assessments in the current study (Adams et al., 2004;Clark & Nowak, 1999;Collins et al., 2017). The area of the gill examined by gross scorers and the histopathologist overlapped but differed which is likely to further reduce agreement (Adams et al., 2004;Taylor, Wynne, Kube, & Elliott, 2007). Lastly, the inclusion of multiple gross scorers with variable experience and the variation of scorers over time, across and within sites, in addition to the use of a scoring system that was new to the scorers, likely introduced variation into the gross score observations unrelated to actual variation in gross pathology (Adams et al., 2004). In conclusion, the overall poor correlation between gross and microscopic lesions is likely explained a combination of factors, from gross scorers, scoring system, histology sampling protocol and the inability to detect mild lesion grossly. However, despite moderate to severe microscopic gill lesions and a high load and prevalence of N. perurans at site G, a concurrent increase in gross score was not recorded for all left arches overall or for the second left gill arch sampled for histology. This observation indicates that cases of moderate to severe gill pathology and AGD may go undetected if gills are only assessed by gross examination.
Cause-specific mortality was assigned as gill-related based on gross examination of dead fish by site staff and/or fish health personnel, information from diagnostic reports and knowledge about environmental and managerial events at the site. Due to the assignment of a single cause and lack of gross examination of each dead fish, there is a risk of underestimating the contribution of infectious diseases to the mortality observed at a site. From human studies of verbal autopsies, it has been shown that it is easier to correctly classify mortality caused by acute, severe trauma, than infectious or cardiovascular disease (Lozano et al., 2011).
Commonly, all mortalities occurring after a handling or treatment event at a site are recorded as treatment-related mortality irrespective of other diseases present in the population or gross findings suggestive of concurrent infectious disease. This may lead to further overestimation of observable management events as causes of mortality and an underestimation of the contribution of infectious and non-infectious disease to the observed mortality. Individual fish dying of diseases in which gross lesions are not readily apparent or non-specific are also more likely to be incorrectly classified. However, despite the weaknesses, cause-specific mortality classification has the advantage of being fast, cheap, can be performed on-site and by site staff and consider data from concurrent laboratory analysis. The resulting data provide an estimate of the drivers of mortality at the population level and can be used for future statistical analysis. But these data were not intended or suited for studies of the interactions between pathogens or interactions between infectious, managerial and environmental factors causing gill disease. Further, the gill-related mortality data should be considered a very conservative estimate of (severe) gill disease in a population.

| Limitations of the study
In addition to the limitations related to the standardized sampling and histopathological assessment discussed previously, there are some limitations of this study related to the fact that this was a field study performed at sites in commercial production. During the study period at sea several fish groups were split or mixed with other fish groups. Mixing leads to a dilution of the study fish group with fish from one or more other pens that may have another disease history. In contrast, the splitting of a fish group is not problematic if groups were randomly split, but unfortunately, whether specific criteria were applied during the splitting of one or more of our fish groups was unknown. The inability to follow the same cohorts throughout the production cycle could have impacted results for the sampling points following the mixing and splitting