Diagnosis of Renibacterium salmoninarum infection in harvested Atlantic salmon (Salmo salar L.) on the east coast of Canada: Clinical findings, sample collection methods and laboratory diagnostic tests.

Chronic subclinical infection with the aetiological agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, presents challenges for the clinical management of disease in farmed salmonids and for prevalence estimation. Harvested salmon sampled at processing plants provide the opportunity to describe subclinical outcomes of BKD using gross necropsy observations and diagnostic test results in farmed Atlantic salmon (Salmo salar L.) populations that are apparently healthy (i.e. alive at harvest) but naturally exposed to R. salmoninarum infection. Sampling of farmed salmon (Population A, n = 124 and Population B, n = 160) was performed immediately post-slaughter as fish were being processed at a plant in New Brunswick, Canada. Populations were selected based on planned harvests from sites with histories of recent exposure events related to clinical BKD as evidenced by the site veterinarian's diagnosis of mortality attributable to BKD: One site (Pop A) had recently increasing mortalities attributed to BKD, and the other site (Pop B) had ongoing low-level mortalities with BKD pathology. As expected with the different exposure histories, Pop A had a higher percentage (57.2%) of R. salmoninarum culture-positive kidney samples compared with similar fish samples in Pop B (17.5%). Diagnosis of R. salmoninarum by gross granulomatous lesions in internal visceral organs, bacterial culture and identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using different swab transport methods, and molecular detection methods (quantitative PCR, qPCR) were compared. Agreement of culture-positive percentages at the sample level was moderate (kappa: 0.61-0.75) among specimens collected using different kidney sampling methods in Pop A and Pop B. The highest proportion of R. salmoninarum-positive cultures occurred when kidney tissues were transported to the laboratory and inoculated directly onto agar using a swab (94% of cultures from Pop A and 82% from Pop B when fish were positive by any culture method). Fish with cumulative lesion scores (severity of granulomatous lesions in 3 different visceral organs) of >4 were all culture positive, and when compared with non-lesioned fish, had substantially higher odds of being culture positive: Pop A: odds ratio (OR) = 73, 95% confidence interval (CI) (7.91, 680.8); Pop B: OR = 66, 95% CI (6.12, 720.7). Our study found that onsite postmortem examinations with severity scores of gross granulomatous lesions were predictive of positive culture results for R. salmoninarum, and they were a useful proxy for assessing prevalence in apparently healthy populations with subclinical infection.

Description of spatiotemporal patterns of infectious salmon anemia virus (ISAV) detections in marine Atlantic Salmon farms in Newfoundland and Labrador.

OBJECTIVE: The objectives of this study were to describe spatiotemporal patterns of infectious salmon anemia virus (ISAV) detections in marine salmonid production sites in the province of Newfoundland and Labrador in Canada. METHODS: Infectious salmon anemia virus surveillance data between 2012 and 2020 from the province of Newfoundland and Labrador were used. Data comprised a total of 94 sampling events from 20 Atlantic Salmon Salmo salar production sites in which ISAV was detected. Using linear regression models, factors influencing time to detection (days from stocking to first ISAV detection) and time to depopulation (days from first detection to production site depopulation) were investigated. RESULT: Based on 28 unique cases, site-level annual incidence risk of ISAV detection ranged from 3% to 29%. The proportion of ISAV detection by PCR in fish samples ranged from 2% to 45% annually. Overall, ISAV variants from the European clade were more common than variants from the North American clade. The type of ISAV clade, detections of ISAV in nearest production sites based on seaway distances, and year of infectious salmon anemia cases were not associated with time to first ISAV detection. Time to depopulation for sites infected with the ISAV-HPRΔ variant was not associated with ISAV North American or European clades. CONCLUSION: Our results contribute to the further understanding of the changing dynamics of infectious salmon anemia detections in Newfoundland and Labrador since its first detection in 2012 and will likely assist in the design of improved disease surveillance and control programs in the province.

Estimating the dispersal of Lepeophtheirus salmonis sea lice within and among Atlantic salmon sites of the Bay of Fundy, New Brunswick.

The objective of this study was to estimate the impact of infestation pressures on the abundance of the parasitic sea louse, Lepeophtheirus salmonis, in the Bay of Fundy, New Brunswick (NB), Canada, using the Fish-iTrends database for the years 2009-2018. Infestation pressures were calculated as time-lagged weighted averages of the abundance of adult female (AF) sea lice within a site (internal infestation pressure: IIP) and among sites (external infestation pressure: EIP). The EIP weights were calculated from seaway distances among sites and a Gaussian kernel density for bandwidths of 5 to 60 km. The EIP with a bandwidth of 10 km had the best fit, as determined with Akaike's information criterion, and historical AF sea lice abundance. This estimated dispersal distance of 10 km was similar to previous studies in Norway, Scotland and in New Brunswick. The infestation pressures estimated from empirical AF sea lice abundance within and among sites significantly increased the abundance of AF sea lice (p < .001). This study concludes that sea lice burdens within Atlantic salmon farms in the Bay of Fundy, NB, are affected by within site management and could be improved by synchronizing treatments between sites.

Production of red tilapia (Oreochromis spp.) in floating cages in the Mekong Delta, Vietnam: mortality and health management.

The Mekong Delta in Vietnam is one of the most productive aquaculture regions in the world, in which the red tilapia (Oreochromis spp.) industry is a small-scale industry that mainly supplies local markets in the delta region. Little is known about the frequency of mortality events and health management in this sector. We describe red tilapia floating cage production systems in the Mekong Delta, Vietnam, for the purposes of quantifying mortality and associated production factors, and describing practices that may influence pathogen introduction and spread to and from farms. In July 2014, approximately 50 red tilapia farmers from 4 provinces (201 farmers in total) were randomly selected and interviewed. Median overall perceived mortality (PM) within a production cycle was 35%. Overall PM was found to be affected by province (p < 0.01), age of farmers (p = 0.01), anticipated main reason for PM in the first 2 wk (p = 0.03), most common market for the fish (p = 0.02), and whether farmers recorded stocking information (p = 0.01). Based on the interviews, we describe and discuss processes that potentially affect pathogen introduction and spread on these farms, such as movements of live and dead fish, distances between farms, mechanical transmission, and biosecurity practices such as treating fish before stocking, using disinfectants, and sharing equipment, and harvesters' movements. This study provides fundamental understanding of red tilapia aquaculture management in the Mekong Delta, and describes management factors that could become important in the event of disease outbreaks.

Effectiveness of emamectin benzoate for treatment of Lepeophtheirus salmonis on farmed Atlantic salmon Salmo salar in the Bay of Fundy, Canada.

Emamectin benzoate (an avermectin chemotherapeutant administered to fish as an in-feed treatment) has been used to treat infestations of sea lice Lepeophtheirus salmonis on farmed Atlantic salmon Salmo salar in the Bay of Fundy, New Brunswick, Canada, since 1999. This retrospective study examined the effectiveness of 114 emamectin benzoate treatment episodes from 2004 to 2008 across 54 farms. Study objectives were to establish whether changes in the effectiveness of emamectin benzoate were present for this period, examine factors associated with treatment outcome, and determine variables that influenced differences in L. salmonis abundance after treatment. The analysis was carried out in 2 parts: first, trends in treatment effectiveness and L. salmonis abundance were explored, and second, statistical modelling (linear and logistic regression) was used to examine the effects of multiple variables on post-treatment abundance and treatment outcome. Post-treatment sea lice abundance increased in the later years examined. Mean abundance differed between locations in the Bay of Fundy, and higher numbers were found at farms closer to the mainland and lower levels were found in the areas around Grand Manan Island. Treatment effectiveness varied by geographical region and decreased over time. There was an increased risk for unsuccessful treatments in 2008, and treatments applied during autumn months were more likely to be ineffective than those applied during summer months.

Optimization and field use of a bioassay to monitor sea lice Lepeophtheirus salmonis sensitivity to emamectin benzoate.

A bioassay for sea lice Lepeophtheirus salmonis sensitivity towards emamectin benzoate (EMB) was validated for field use. A probit regression model with natural responsiveness was used for the number of affected (moribund or dead) sea lice in bioassays involving different concentrations of EMB. Bioassay optimization included an evaluation of the inter-rater reliability of sea lice responsiveness to EMB and an evaluation of gender-related differences in susceptibility. Adoption of a set of bioassay response criteria improved the concordance (evaluated using the concordance correlation coefficient) between raters' assessments and the model estimation of EC50 values (the 'effective concentration' leading to a response of 50% of the lice not prone to natural response). An evaluation of gender-related differences in EMB susceptibility indicated that preadult stage female sea lice exhibited a significantly larger sensitivity towards EMB in 12 of 19 bioassays compared to preadult males. In order to evaluate sea lice sensitivity to EMB in eastern Canada, the intensive salmon farming area in the Bay of Fundy in southwestern New Brunswick was divided into 4 distinct regions based on industry health management practices and hydrographics. A total of 38 bioassays were completed from 2002 to 2005 using populations of preadult stage sea lice collected from Atlantic salmon Salmo salar farms within the 4 described regions. There was no significant overall effect of region or year on EC50 values; however, analysis of variance indicated a significant effect of time of year on EC50 values in 2002 and a potential effect in 2004 to 2005. Although the range of EC50 values obtained in this 3 yr study did not appear sufficient to affect current clinical success in the control of sea lice, the results suggest a seasonal- or temperature-associated variation in sensitivity to EMB. This will need to be considered if changes in EMB efficacy occur in the future.

Heart and skeletal muscle inflammation (HSMI) disease diagnosed on a British Columbia salmon farm through a longitudinal farm study.

Heart and skeletal muscle inflammation (HSMI) is an emerging disease of marine-farmed Atlantic Salmon (Salmo salar), first recognized in 1999 in Norway, and later also reported in Scotland and Chile. We undertook a longitudinal study involving health evaluation over an entire marine production cycle on one salmon farm in British Columbia (Canada). In previous production cycles at this farm site and others in the vicinity, cardiac lesions not linked to a specific infectious agent or disease were identified. Histologic assessments of both live and moribund fish samples collected at the farm during the longitudinal study documented at the population level the development, peak, and recovery phases of HSMI. The fish underwent histopathological evaluation of all tissues, Twort's Gram staining, immunohistochemistry, and molecular quantification in heart tissue of 44 agents known or suspected to cause disease in salmon. Our analysis showed evidence of HSMI histopathological lesions over an 11-month timespan, with the prevalence of lesions peaking at 80-100% in sampled fish, despite mild clinical signs with no associated elevation in mortalities reported at the farm level. Diffuse mononuclear inflammation and myodegeneration, consistent with HSMI, was the predominant histologic observation in affected heart and skeletal muscle. Infective agent monitoring identified three agents at high prevalence in salmon heart tissue, including Piscine orthoreovirus (PRV), and parasites Paranucleospora theridion and Kudoa thyrsites. However, PRV alone was statistically correlated with the occurrence and severity of histopathological lesions in the heart. Immunohistochemical staining further localized PRV throughout HSMI development, with the virus found mainly within red blood cells in early cases, moving into the cardiomyocytes within or, more often, on the periphery of the inflammatory reaction during the peak disease, and reducing to low or undetectable levels later in the production cycle. This study represents the first longitudinal assessment of HSMI in a salmon farm in British Columbia, providing new insights on the pathogenesis of the disease.

Risk factors for outbreaks of infectious salmon anemia in farmed Atlantic salmon, Salmo salar.

Infectious salmon anemia (ISA) is a viral disease occurring in farmed Atlantic salmon (Salmo salar) that is characterized by lethargy, anorexia, anemia and death. To control the disease in New Brunswick, Canada, 7.5 million fish from outbreak cages have been destroyed since 1997. Despite changes made by farmers, 2002 was the worst year ever for ISA losses in the region. We evaluated the associations between potential risk factors and ISA outbreaks in the Atlantic-salmon sites in New Brunswick. This was a multilevel study in which the site-level design was a retrospective cohort study while the cage-level design was a modified case-cohort study. The questionnaire was divided into site-level questions, cage-level questions and hatchery information. The important factors identified by this study can be categorized as environmental, farmer controlled or industry controlled according to the capacity to change or eliminate them. Environmental risk factors such as increasing the depth of the net (if nets were 3m, OR=3.34) are for the most part dictated by site location. Wild pollock (Pollachius virens) in the cage reflects the number of wild pollock that live in the site location. If there were >or=1000 pollock in the cage, the odds of disease in the cage increased 4.43-fold. Risk factors that are under farm control include increasing the number of times that the salmon are treated for sea lice (OR=3.31 if lice treatments are 99 g) and improving on the adaptation of smolts to seawater to reduce post-transfer mortalities (OR=4.52 if there was at least one cage with post-transfer mortalities >5%). The industry-controlled factors need to be addressed by the industry as a whole. Organizing boat travel to minimize the time and frequency of boats travelling to or by sites currently is being reviewed. This will be extremely important because the OR=9.43 if processing boats travel within 1 km of the site and the OR=4.03 if the site has dry feed delivered by the feed company. Because the hazard ratio increased stepwise from 1 if the nearest neighbor with ISA was >or=5 km up to 5.5 if the nearest site with ISA was within 0.5 km, increasing the distance between sites might be necessary for effective control.

Application of surveillance data in evaluation of diagnostic tests for infectious salmon anemia.

Infectious salmon anemia (ISA) is a viral disease in farmed Atlantic salmon Salmo salar, characterized by lethargy, anorexia, anemia and death. Test methods used for regulatory decisions to remove infected cages include the indirect fluorescent antibody test (IFAT), the reverse transcription-polymerase chain reaction test (RT-PCR), and virus isolation (VI). However, no thorough evaluation of these diagnostic tests has been carried out on field samples. The objective of this study was to evaluate the sensitivity and specificity of ISA diagnostic tests as individual tests and in combinations, using data collected by the provincial government surveillance program. Because a 'gold standard' reference test for ISA was not available, cage status was based on clinical disease records. A pool of fish from negative farms that had never had clinical ISA and a pool of fish from positive cages that were experiencing an outbreak of clinical ISA were obtained and assumed to be uninfected and infected respectively. A total of 1071 fish were used in this study. Depending on the test's cut-off value, the sensitivity and specificity for histopathology ranged from 30 to 73 and 72 to 99% respectively. IFAT had sensitivities and specificities in the range of 64 to 83 and 96 to 100% respectively. For the RT-PCR, sensitivity and specificity were 93 and 98% respectively. Test performances were also evaluated in series and in parallel combinations. Sensitivities are maximized when tests are evaluated in parallel, and ranged from 75 to 98%. Specificities are maximized when the tests are evaluated in series, and ranged from 99 to 100%. Current surveillance testing protocols should be reviewed to capitalize on this newly available information on test characteristics.

Lack of evidence of infectious salmon anemia virus in pollock Pollachius virens cohabitating with infected farmed Atlantic salmon Salmo salar.

The infectious salmon anemia (ISA) virus causes lethargy, anemia, hemorrhage of the internal organs, and death in farmed Atlantic salmon Salmo salar. It has been a cause of disease in Norwegian farmed Atlantic salmon since 1984 and has since been identified in Canada, Scotland, the United States, and the Faroe Islands. Wild fish have been proposed as a viral reservoir because they are capable of close contact with farmed salmon. Laboratory studies have shown that brown trout and sea trout Salmo trutta, rainbow trout Oncorhynchus mykiss, and herring Clupea harengus tested positive for the virus weeks after intra-peritoneal injection of the ISA virus. Pollock Pollachius virens are commonly found in and around salmon cages, and their close association with the salmon makes them an important potential viral reservoir to consider. The objective of this study was to determine the presence or prevalence of ISA virus in pollock cohabitating with ISA-infected farmed Atlantic salmon. Kidney tissue from 93 pollock that were living with ISA-infected salmon in sea cages were tested with reverse transcription-polymerase chain reaction (RT-PCR) test. Results yielded the expected 193 bp product for positive controls, while no product was observed in any of the pollock samples, resulting in an ISA viral prevalence of 0%. This study strengthens the evidence that pollock are unlikely to be an ISA virus reservoir for farmed Atlantic salmon.

Evidence for occurrence of an organophosphate-resistant type of acetylcholinesterase in strains of sea lice (Lepeophtheirus salmonis Krøyer).

Acetylcholinesterase (AChE) is the target of a major pesticide family, the organophosphates, which were extensively used as control agents of sea lice on farmed salmonids in the early 1990s. From the mid-1990s the organophosphates dichlorvos and azamethiphos were seriously compromised by the development of resistance. AChE insensitive to organophosphate chemotherapeutants has been identified as a major resistance mechanism in numerous arthropod species, and in this study, target-site resistance was confirmed in the crustacean Lepeophtheirus salmonis Krøyer isolated from several fish-farming areas in Norway and Canada. A bimolecular rate assay demonstrated the presence of two AChE enzymes with different sensitivities towards azamethiphos, one that was rapidly inactivated and one that was very slowly inactivated. To our knowledge this is the first report of target-site resistance towards organophosphates in a third class of arthropods, the Crustacea.

Selection of a cutoff value for real-time polymerase chain reaction results to fit a diagnostic purpose: analytical and epidemiologic approaches.

Diagnostic laboratories frequently select a subjective cutoff value for real-time amplification assays, above which a threshold cycle (Ct) value is deemed false. Commonly, higher Ct values are interpreted as amplification or fluorescence artifacts, or cross contaminations. Although the implementation of Ct cutoff might be reasonable, its justification and selection should be based on evidence. The current article reviewed evidence-based strategies to select Ct cutoffs grouped in analytical and epidemiologic approaches. Analytical strategies use criteria gathered during the assay development and include fluorescence threshold, reaction end-cycle, limit of detection, and artifact investigation. Variability in amplification efficacy across test runs may induce some instability in an intended Ct cutoff and requires some standardization or normalization procedures. Epidemiologic strategies use criteria based on either the probability or the cost of a false test result associated with a specified cutoff. Cutoffs, depending on the intended purpose of the test, can be selected graphically to minimize the probability of either false-positive or false-negative results by using two-graph receiver operating characteristics curves. The assay's diagnostic sensitivity and specificity may vary with the tested population, thus, the estimated two-graph receiver operating characteristics curve is population dependent and should be established for the targeted population. Although the selection of a cutoff based on misclassification cost depends on infection prevalence, the selection based on predictive values does not. To optimize the test average diagnostic performance, the Ct cutoff should be selected when diagnostic odds ratio is maximal. Epidemiologic approaches were illustrated by selecting Ct cutoffs for a real-time assay for Infectious salmon anemia virus.