Descriptive epidemiology of variants of infectious salmon anaemia virus in four Atlantic salmon farms in Newfoundland and Labrador, Canada.

An incursion of infectious salmon anaemia virus (ISAV) was detected in 2020 in southern Newfoundland, Canada. This resulted in an outbreak affecting four marine farms stocking Atlantic salmon (Salmo salar L.) vaccinated against ISAV. This study provides the first description of epidemiologic characteristics of an ISAV outbreak in 2020 and 2021, and detected ISAV variants at the population level. Fish kidneys were screened for ISAV by real-time RT-PCR and non-negative samples were submitted for genotyping and further diagnostic testing. Nine distinct ISAV variants were identified: five European and three North American (NA) HPRΔ ISAV, and one NA-HPR0 ISAV variant. A notable finding was the concurrent detection of both an HPR0 and an HPRΔ ISAV variant in one individual fish. In two farms, both European and NA variants were simultaneously detected, while in the other two farms either NA or European variants were identified, but not both together. Generally, mortality increases followed rises in ISAV prevalence and cycle threshold values on RT-PCR decreased with time. Epidemiologic descriptions of ISAV outbreaks in Atlantic Canada contributes to the understanding of local disease dynamics and identification of changes thereof. Such insights are essential for the strengthening of disease management plans.

Estimating sea lice infestation pressure on salmon farms: Comparing different methods using multivariate state-space models.

Sea lice are ectoparasites of salmonids, and are considered to be one of the main threats to Atlantic salmon farming. Sea lice infestation on a farm is usually initiated by attachment of the free-living copepodid stage derived from the surrounding water, frequently originating from adult lice on the same farm or from neighboring salmonid farms, referred to as internal and external sources, respectively. Various approaches have been proposed to quantify sea lice infestation pressure on farms to improve the management of this pest. Here, we review and compare five of these methods based on sea lice data from 20 farms located near Grand Manan island in the Bay of Fundy, New Brunswick, Canada. Internal and external infestation pressures (IIP and EIP, respectively) were estimated using different approaches, and their effects were modeled either by a unique parameter for all production cycles or by different parameters for each production cycle, using a multivariate state-space model. Predictive variables, such as water temperature and sea lice treatments, were included in the model, and their effects across production cycles were estimated along with those of other model parameters. Results showed that models with only EIP explained the variation in the data better than models with only IIP, and that models with unique IIP and unique EIP for all cycles were generally associated with the best model fit. The simplest, fixed lag method for calculating infestation pressure had the best predictive performance in our models among the methods studied.

A modelling approach to predict the variation of repeatability and reproducibility of a RT-PCR assay for infectious salmon anaemia virus across infection prevalences and infection stages.

Conventional studies on the precision of diagnostic tests with binary outcomes report single descriptive estimates of agreement for a particular pool of samples. However, agreement for binary tests is intrinsically associated with the assay operating characteristics that are influenced by population and laboratory covariate factors. Therefore, reporting agreement estimates under various conditions may be more appropriate for diagnostic test comprehension. In this study, the influence of various submission factors (tissue sample homogenization, prevalence of infection and pathogen level) on agreement was further analyzed using test result information from a previous descriptive report of within and between laboratories agreement (repeatability and reproducibility, respectively) of a Reverse-Transcription Polymerase Chain Reaction (RT-PCR) assay for infectious salmon anaemia virus (ISAV). Multilevel logistic regression models were constructed separately for non-, low- or high-infected salmon (classified using a study pseudogold standard) to predict probabilities of testing positive under different testing conditions. For each of the 3 infection categories, agreement and kappa values within and between laboratories were computed from the models' predicted values using probability formulae. Thereafter, overall estimates were predicted using simple category weighting for various proportions of infection stages. Agreement varied substantially among infection categories and, consequently, overall repeatability and reproducibility varied greatly with prevalence. This confirmed that the report of a single descriptive estimate (corresponding to a set prevalence) may not be appropriate. Low-infected fish had the lowest agreement estimate which was improved by sample homogenization. This supported a heterogeneous distribution of ISAV in early infected salmon kidney. However, tissue homogenization increased the probability to obtain a false-positive test result (cross-contamination suspected) and decreased agreement in non-infected fish. Compared to conventional report of test agreement estimation, the modelling approach identified influencing submission factors and provided predictive intervals of agreement that give a better expectation and understanding of assay repeatability and reproducibility under different circumstances of use.

Use of a third class in latent class modelling for the diagnostic evaluation of five infectious salmon anaemia virus detection tests.

In the absence of a reference standard, a latent class model (LCM) was used in this study to assess diagnostic sensitivity (DSe) and specificity (DSp) of a recently developed reverse-transcription polymerase chain reaction (RT-PCR) for infectious salmon anaemia virus (ISAV). The study included 4 populations of Atlantic salmon, and to ensure the identifiability of the LCM, four additional detection methods were used in parallel including real-time RT-PCR (qRT-PCR), virus isolation (VI), indirect fluorescent antibody test (IFAT), and a lateral flow immunoassay (LFI). While a conventional LCM assumes DSe and DSp to be constant across the populations, Nérette et al. (2008) previously reported concerns about non-constant DSp of RT-PCR, which detects viral RNA from both active and inactive viral particles. It was suspected that some ISAV recovered fish may carry residual RNA and may be more likely to test positive compared to naïve fish. The various mixture distributions of the two sub-classes of non-infected fish would lead to a non-constant combined DSp estimate across populations. Within a Bayesian framework, the conventional two-class LCM was extended to three classes of infection stages (naïve non-infected, recovered non-infected carrying RNA, and infected). The resulting analysis confirmed the existence of three classes of fish with substantially different test performances for ISAV. For infected fish, DSe of RT-PCRs and VI approximated 90%, and antibody based assays were the least sensitive (DSe around 65%). Regardless of the test, the DSp estimates on naïve fish were all above 98% with LFI being in average the most specific. Only RT-PCR and qRT-PCR tested positive with the additional class of recovered fish (DSp around 30%). The true infectious status of this sub-class (i.e. viral RNA carriers) is debatable and requires further knowledge about ISAV infection dynamics at the fish level. Promising applications of multiple class estimates require adjustments of traditional test interpretation and further epidemiological knowledge of the infection dynamics at the population level.

Traditional descriptive analysis and novel visual representation of diagnostic repeatability and reproducibility: application to an infectious salmon anaemia virus RT-PCR assay.

As a component of diagnostic test evaluation, the estimation of repeatability and reproducibility of an assay is necessary to assess the robustness and the transferability of the method among laboratories. Respectively defined as the agreement within and between laboratories, repeatability and reproducibility of a qualitative diagnostic test are traditionally reported using observed proportion of agreement or Kappa values. Applied to a recently designed RT-PCR assay for the detection of infectious salmon anaemia virus, repeatability only within a national reference laboratory and reproducibility with two additional independent regional laboratories were investigated. Homogenization of fish kidney tissue was conducted to potentially provide more uniform submission material, and to assess the effect of homogenization on laboratory comparability. Comparison of agreement between non-homogenized and homogenized tissue samples revealed different patterns of test results and unexpected alterations of agreement due to homogenization. This observation may be explained by cross-contamination of some samples during the homogenization process. One of the laboratories was in clear disagreement with the two others and impacted the overall reproducibility of the assay. Agreement levels were visually described using a novel tree-shape representation inspired from phylogenetic studies. The resulting phylogram illustrated the proximity of test findings between repeated samples within a laboratory and between laboratories, and facilitated the interpretation of the agreement levels.

Evaluation of a reverse transcriptase polymerase chain reaction test and virus isolation on field samples collected for the diagnosis of infectious hematopoietic necrosis virus in cultured Atlantic salmon in British Columbia.

Infectious hematopoietic necrosis virus (IHNV) has been found to cause disease in cultured salmon of the Pacific Northwest region of North America. Diagnosis of IHNV by virus isolation (VI) can take over 2 weeks. Recently, a rapid reverse transcriptase (RT) polymerase chain reaction (PCR) test on fish tissues has been used for diagnosis. Test performances of the VI and RT PCR assays were compared using samples collected in the field. The effect of different storage conditions (tissue frozen with or without RNAlater [Ambion, Inc., Austin, Texas] versus fresh tissue) on the diagnostic tests was also evaluated. Based on the limited number of samples tested, the operating characteristics of RT PCR were very similar to those of VI; therefore, this method is likely suitable for testing field samples for IHNV. The ability of the tests to identify a positive fish ranged from 74% to 89%. Freezing samples at -80 degrees C before testing did not negatively affect the performance of RT PCR or VI. However, due to reduced test performance, RNAlater frozen storage is not recommended without further investigation.

Using pseudogold standards and latent-class analysis in combination to evaluate the accuracy of three diagnostic tests.

We previously reported our use of latent-class models to estimate the sensitivity (Se) and specificity (Sp) for each of three tests used to monitor farmed salmon for infectious salmon anaemia virus (ISAv). Those tests were reverse-transcriptase polymerase chain reaction (RT-PCR), virus isolation (VI), and an indirect immunofluorescent-antibody test (IFAT). We used tissues from 403 salmon from four populations presumed to have different prevalence of ISAv. However, no formal evaluation of the assumptions of conditional independence and constant accuracy had been carried out. In our present study, we "adjusted" that and used two pseudogold standards (a composite reference standard and a study pseudogold), as indicative of the true health status of each fish. The assumption of constant accuracy across populations was evaluated using separate random-effects logistic-regression models for fish classified as D+ or D- (disease positive or negative, according to the pseudogold standards) with study population included in the model to determine if it affected the probability of a positive test result. Where there was evidence of variation in test accuracy across populations, the issue was further investigated using separate latent-class models with informative priors for each study population. Our results suggested that only one PCR test had an accuracy that varied across populations. The assumption of conditional independence among tests was first evaluated using log-linear models of D+ and D- fish with significant interaction between test results indicative of conditional dependence. Latent-class models which incorporated up to two pairs of between-test dependencies were also fit using Bayesian methods. The two approaches showed considerable evidence of dependence between IFAT and VI and some evidence of dependence between one PCR and IFAT. Results obtained from both maximum-likelihood models and from Bayesian analyses of models allowing for conditional dependence between two pairs of tests were consistent with those obtained with the pseudogold standards. The results suggest that pseudogold standards can help in choosing a correct dependence structure and should be used in combination with latent-class models.

Spinal deformities in farmed Atlantic salmon.

Spinal deformities in farmed Atlantic salmon (Salmo salar) are often observed in intensive farming systems and result in production losses. Many putative factors have been implicated with the formation of spinal deformities in larger salmon. This condition has been described as broken back syndrome, curvy back disease, and short tails.