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.

Development and validation of main spectral profile for rapid identification of Yersinia ruckeri isolated from Atlantic salmon using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) allows rapid and reliable identification of microorganisms. The accuracy of bacterial identification using MALDI-TOF MS depends on main spectral profiles (MSPs) provided in a quality-assured commercial reference library, which requires ongoing improvement. This study aimed to develop and validate an in-house MALDI-TOF MS MSP to rapidly identify Yersinia ruckeri isolated from Atlantic salmon (Salmo salar). The novel MSP was prepared using an isolate of Y. ruckeri recovered from Atlantic salmon and confirmed by 16S rRNA gene sequencing. Subsequently, a validation set which comprises 29 isolates of Y. ruckeri were examined from three fishes: Atlantic salmon (Salmo salar) (n = 26), American eel (Anguilla rostrata) (n = 1), and Atlantic cod (Gadus morhua) (n = 2). These isolates were randomly selected from the Atlantic Veterinary College, Aquatic Diagnostic Services Bacteriology Laboratory's culture collection to validate the novel MSP. Analytical sensitivity of MALDI-TOF MS using the novel MSP to identify the validation set was 86.2%. Repeatability was assessed by acquiring spectra from 30 different spots of a randomly-selected isolate of Y. ruckeri, and analyzed spectra from each spot were compared against the novel MSP. The coefficient of variation was 3.3%. The novel MSP clustered with Bruker MSPs (n = 3) of Y. ruckeri in the reference library and did not falsely identify any closely related bacteria to Y. ruckeri. This study reports the development of a novel MSP of high analytical sensitivity and specificity for rapid identification of Y. ruckeri using MALDI-TOF MS.

Performance characteristics of two real-time TaqMan polymerase chain reaction assays for the detection of WSSV in clinically diseased and apparently healthy prawns.

This study aimed to generate data on performance characteristics for 2 real-time TaqMan PCR assays (CSIRO and WOAH WSSV qPCRs) for the purposes of (1) detection of white spot syndrome virus (WSSV) in clinically diseased prawns and (2) detection of WSSV in apparently healthy prawns. Analytical sensitivity of both assays was 2 to 20 genome copies per reaction, and analytical specificity was 100% after testing nucleic acid from 9 heterologous prawn pathogens and 4 prawn species. Results obtained after testing more than 20 000 samples in up to 559 runs with the CSIRO WSSV qPCR and up to 293 runs with the WOAH WSSV qPCR demonstrated satisfactory repeatability for both assays. Both assays demonstrated median diagnostic sensitivity (DSe) 100% (95% CI: 94.9-100%) when testing clinically diseased prawns. When 1591 test results from apparently healthy prawns were analysed by Bayesian latent class analysis, median DSe and diagnostic specificity (DSp) were 82.9% (95% probability interval [PI]: 75.0-90.2%) and 99.7% (95% PI: 98.6-99.99%) for the CSIRO WSSV qPCR and 76.8% (95% PI: 68.9-84.9%) and 99.7% (95% PI: 98.7-99.99%) for the WOAH WSSV qPCR. When both assays were interpreted in parallel, median DSe increased to 98.3 (95% PI: 91.6-99.99%), and median DSp decreased slightly to 99.4% (95% PI: 97.9-99.99%). Routine testing of quantified positive controls by laboratories in the Australian laboratory network demonstrated satisfactory reproducibility of the CSIRO WSSV qPCR assay. Both assays demonstrated comparable performance characteristics, and the results contribute to the validation data required in the WOAH validation pathway for the purposes of detection of WSSV in clinically diseased and apparently healthy prawns.

Bayesian estimation of sensitivity and specificity of a rapid mastitis test kit, bacterial culture, and PCR for detection of Staphylococcus aureus, Streptococcus species, and coliforms in bovine milk samples.

Our objectives were to evaluate the diagnostic accuracy of a rapid and novel immunochromatography-based mastitis kit that includes 3 independent tests to detect coliforms (Escherichia coli or Klebsiella pneumoniae), Streptococcus spp., and Staphylococcus aureus. The kit was developed to facilitate diagnostic-based mastitis treatment. Validation of the kit was based on 154 aseptically collected mastitis samples from 2 clinical herds (clinical population) and 120 milk samples from 3 nonclinical herds (nonclinical population) without clinical cases at the time of enrollment. One herd sampled at different times was common to both populations. A 3-test in 2-population Bayesian latent class model with uniform priors for all test parameters except specificity of culture, which was modeled informatively, was used to estimate sensitivity (Se) and specificity (Sp) of the test kit, culture, and PCR at the cow level. The mastitis test kit's 96.9% Sp for Streptococcus spp. had a low false positive percentage (3.1%), which, together with the kit's rapid turnaround time for results, makes it a suitable initial screening test that producers can use to identify clinical cows to treat based on Streptococcus spp. mastitis in kit-positive results. Due to the 60.4% kit Se, producers should follow up on Streptococcus spp. kit-negative cows using a confirmatory test such as PCR (Sp of 98.4%) or culture (Sp of 99.6%). In contrast, aerobic culture had Se of 76.5% and Sp of 99.6% for Streptococcus spp. Similarly, the Sp of the kit (98.2%) and culture (99.8%) for Staph. aureus were particularly high, and even though the kit's Se (61.0%) was lower than culture (88.4%; posterior probability of difference 98%), the kit could be beneficial before use of a confirmatory test for kit-negative samples due to its ease and rapid turnaround time. Mostly, quantitative real-time (q)PCR outperformed the kit's Se (37.7%) and Sp (92.9%) for coliforms, as well as the kit's Se (60.4%) for Streptococcus spp. However, qPCR may require more technical skills and turnaround time for final results. Use of the on-farm mastitis test kit evaluated in the present study could enhance sustainable antimicrobial drug use by rapidly identifying Streptococcus mastitis for targeted treatment. Furthermore, the kit may be used in a Staph. aureus outbreak where cows can be rapidly screened to identify cases for segregation or culling during an outbreak and kit-negative cows further confirmed by milk culture or qPCR. However, the cost-effectiveness of such an approach has not been investigated.

Antimicrobial Susceptibility Profiles of Bacteria Commonly Isolated from Farmed Salmonids in Atlantic Canada (2000-2021).

Bacterial infection and antimicrobial resistance are important constraints in the production and sustainability of farmed salmonids. This retrospective study aimed to describe the frequency of bacterial isolates and antimicrobial resistance profiles in salmonid aquaculture in Atlantic Canada. Bacterial isolates and antimicrobial susceptibility testing (AST) results assessed by disk diffusion testing were summarized for 18,776 Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) samples from 2291 unique cases submitted to the Atlantic Veterinary College, Aquatic Diagnostic Services Bacteriology Laboratory from 2000 to 2021. Kidney was the most commonly submitted tissue (60.29%, n = 11,320), and these specimens were mostly submitted as swabs (63.68%, n = 11,957). The most prevalent pathogens detected in these cases were Yersinia ruckeri type 1 (5.54%, n = 127), Renibacterium salmoninarum (2.10%, n = 48), Aeromonas salmonicida (atypical) (1.66%, n = 38), and Pseudomonas fluorescens (1.22%, n = 28). Most bacterial isolates tested (n = 918) showed resistance to florfenicol, oxytetracycline, ormetoprim-sulfadimethoxine, and trimethoprim-sulfamethoxazole, but not to enrofloxacin. This report provides baseline data for antimicrobial surveillance programs that investigate emerging antimicrobial resistance trends in salmonid aquaculture in Atlantic Canada.

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.

Cost-effectiveness of longitudinal surveillance for Piscirickettsia salmonis using qPCR in Atlantic salmon farms (Salmo salar) in Chile.

Costs of diagnostic testing including sample collection, sampling frequency and sample size are an important consideration in the evaluation of the economic feasibility of alternative surveillance strategies for detection of infectious diseases in aquatic animals. In Chile, Piscirickettsia salmonis is the primary reason for antibiotic treatments in farmed Atlantic salmon. In 2012, a surveillance and control programme for piscirickettsiosis was established with an overall goal of reducing antibiotic use. The present study estimated the cost-effectiveness of different sampling frequencies and sample sizes to achieve at least 95% confidence of early detection of P. salmonis at the netpen and farm levels using a validated qPCR test. We developed a stochastic model that incorporated variability in test accuracy, within-pen prevalence and sampling costs. Our findings indicated that the current piscirickettsiosis surveillance programme based on risk-based sampling of five moribund or dead fish from 2 to 3 netpens is cost-effective and gives a high probability of detection of P. salmonis in Atlantic salmon farms in Chile at both the netpen and farm levels. Results from this study should incentivize salmon farmers to establish cost-effective strategies for early detection of P. salmonis infection and the application of this approach to other highly infectious diseases.

Diagnostic sensitivity of pooled samples for the detection of tilapia lake virus and application to the estimation of within-farm prevalence.

Tilapia lake virus (TiLV) is a highly contagious novel orthomyxo-like RNA virus that is negatively impacting tilapia production worldwide. To prevent TiLV from spreading globally, the infection status of source farms needs to be established prior to the movement of live tilapia to minimize the risk of horizontal transmission. However, testing individual fish for TiLV requires large sample sizes, when within-farm prevalence is low and is costly, time-consuming, and labour-intensive. The objective of the present study was to evaluate the use of pool testing for TiLV detection and to estimate within-farm prevalence based on the percentage of positive pooled samples. Pooled samples of liver and spleen were prepared by diluting different numbers of positive tissue samples with negative homogenate tissue samples. A tissue pool from 5 or 10 individual fish containing at least one TiLV-positive sample was sufficient to yield a positive result except when cycle threshold (Ct) values were between 31 and the cut-off value of 34. Additionally, our study characterized viral load in two farms after TiLV outbreaks. Bayesian modelling showed that within-farm prevalence could be estimated from the percentage of positive pools of size 5 using prior information about pool sensitivity and specificity, and prevalence, and assuming random sampling of tilapia from infected ponds. Ninety-five percent posterior intervals for prevalence were slightly wider than those obtained based on the results of individual samples. Findings in the present study corroborate the use of a pooling strategy for post-outbreak surveillance of TiLV.

Bayesian estimation of diagnostic sensitivity and specificity of a qPCR and a bacteriological culture method for Piscirickettsia salmonis in farmed Atlantic salmon (Salmo salar L.) in Chile.

Early detection of piscirickettsiosis is an important purpose of government- and industry-based surveillance for the disease in Atlantic salmon farms in Chile. Real-time qPCRs are currently used for surveillance because bacterial isolation is inadequately sensitive or rapid enough for routine use. Since no perfect tests exist, we used Bayesian latent class models to estimate diagnostic sensitivity (DSe) and specificity (DSp) of qPCR and culture using separate two-test, single-population models for three farms (n = 148, 151, 44). Informative priors were used for DSp (culture (beta(999,1); qPCR (beta(98,2)), and flat priors (beta 1,1) for DSe and prevalence. Models were run for liver and kidney tissues combined and separately, based on the presence of selected gross-pathological signs. Across all models, qPCR DSe was 5- to 30-fold greater than for culture. Combined-tissue qPCR median DSe was highest in Farm 3 (sampled during P. salmonis outbreak (DSe = 97.6%)) versus Farm 1 (DSe = 85.6%) or Farm 2 (DSe = 83.5%), both sampled before clinical disease. Median DSe of qPCR was similar for liver and kidney, but higher when gross-pathological signs were evident at necropsy. High DSe and DSp and rapid turnaround-time indicate that the qPCR is fit for surveillance programmes and diagnosis during an outbreak. Targeted testing of salmon with gross-pathological signs can enhance DSe.

Validation of laboratory tests for infectious diseases in wild mammals: review and recommendations.

Evaluation of the diagnostic sensitivity (DSe) and specificity (DSp) of tests for infectious diseases in wild animals is challenging, and some of the limitations may affect compliance with the OIE-recommended test validation pathway. We conducted a methodologic review of test validation studies for OIE-listed diseases in wild mammals published between 2008 and 2017 and focused on study design, statistical analysis, and reporting of results. Most published papers addressed Mycobacterium bovis infection in one or more wildlife species. Our review revealed limitations or missing information about sampled animals, identification criteria for positive and negative samples (case definition), representativeness of source and target populations, and species in the study, as well as information identifying animals sampled for calculations of DSe and DSp as naturally infected captive, free-ranging, or experimentally challenged animals. The deficiencies may have reflected omissions in reporting rather than design flaws, although lack of random sampling might have induced bias in estimates of DSe and DSp. We used case studies of validation of tests for hemorrhagic diseases in deer and white-nose syndrome in hibernating bats to demonstrate approaches for validation when new pathogen serotypes or genotypes are detected and diagnostic algorithms are changed, and how purposes of tests evolve together with the evolution of the pathogen after identification. We describe potential benefits of experimental challenge studies for obtaining DSe and DSp estimates, methods to maintain sample integrity, and Bayesian latent class models for statistical analysis. We make recommendations for improvements in future studies of detection test accuracy in wild mammals.

Review of infectious agent occurrence in wild salmonids in British Columbia, Canada.

Wild Pacific salmonids (WPS) are economically and culturally important to the Pacific North region. Most recently, some populations of WPS have been in decline. Of hypothesized factors contributing to the decline, infectious agents have been postulated to increase the risk of mortality in Pacific salmon. We present a literature review of both published journal and unpublished data to describe the distribution of infectious agents reported in wild Pacific salmonid populations in British Columbia (BC), Canada. We targeted 10 infectious agents, considered to potentially cause severe economic losses in Atlantic salmon or be of conservation concern for wild salmon in BC. The findings indicated a low frequency of infectious hematopoietic necrosis virus, piscine orthoreovirus, viral haemorrhagic septicaemia virus, Aeromonas salmonicida, Renibacterium salmoninarum, Piscirickettsia salmonis and other Rickettsia-like organisms, Yersinia ruckeri, Tenacibaculum maritimum and Moritella viscosa. No positive results were reported for infestations with Paramoeba perurans in peer-reviewed papers and the DFO Fish Pathology Program database. This review synthesizes existing information, as well as gaps therein, that can support the design and implementation of a long-term surveillance programme of infectious agents in wild salmonids in BC.

To pool or not to pool? Guidelines for pooling samples for use in surveillance testing of infectious diseases in aquatic animals.

Samples from multiple animals may be pooled and tested to reduce costs of surveillance for infectious agents in aquatic animal populations. The primary advantage of pooling is increased population-level coverage when prevalence is low (<10%) and the number of tests is fixed, because of increased likelihood of including target analyte from at least one infected animal in a tested pool. Important questions and a priori design considerations need to be addressed. Unfortunately, pooling recommendations in disease-specific chapters of the 2018 OIE Aquatic Manual are incomplete and, except for amphibian chytrid fungus, are not supported by peer-reviewed research. A systematic review identified only 12 peer-reviewed aquatic diagnostic accuracy and surveillance studies using pooled samples. No clear patterns for pooling methods and characteristics were evident across reviewed studies, although most authors agreed there is a negative effect on detection. Therefore, our purpose was to review pooling procedures used in published aquatic infectious disease research, present evidence-based guidelines, and provide simulated data examples for white spot syndrome virus in shrimp. A decision tree of pooling guidelines was developed for use by peer-reviewed journals and research institutions for the design, statistical analysis and reporting of comparative accuracy studies of individual and pooled tests for surveillance purposes.

Gold standards are out and Bayes is in: Implementing the cure for imperfect reference tests in diagnostic accuracy studies.

Bayesian mixture models, often termed latent class models, allow users to estimate the diagnostic accuracy of tests and true prevalence in one or more populations when the positive and/or negative reference standards are imperfect. Moreover, they allow the data analyst to show the superiority of a novel test over an old test, even if this old test is the (imperfect) reference standard. We use published data on Toxoplasmosis in pigs to explore the effects of numbers of tests, numbers of populations, and dependence structure among tests to ensure model (local) identifiability. We discuss and make recommendations about use of priors, sensitivity analysis, model identifiability and study design options, and strongly argue for the use of Bayesian mixture models as a logical and coherent approach for estimating the diagnostic accuracy of two or more tests.

Economic appraisal of vaccination against Streptoccocus agalactiae in Nile tilapia farms in Brazil.

Infection with Streptococcus agalactiae causes mortality and major economic losses in Nile tilapia (Oreochromis niloticus) farming worldwide. In Brazil, serotype strains Ia, Ib and III have been isolated in streptococcosis outbreaks, but serotype Ib is the most prevalent. Vaccination is considered an effective method to prevent economically-important diseases in aquaculture and has been associated with decreased use of antibiotics and improvements in fish survival. We developed a flexible partial-budget model to undertake an economic appraisal of vaccination against Streptococcus agalactiae in Nile tilapia farmed in net cages in large reservoirs. The model considers the benefits and costs that are likely to be associated with vaccination at the farm-level, in one production cycle. We built three epidemiological scenarios of cumulative mortality attributable to S. agalactiae (5%, 10%, and 20%, per production cycle) in a non-vaccinated farm. For each scenario, we applied a stochastic model to simulate the net return of vaccination, given a combination of values of "vaccine efficacy", "gain in feed conversion ratio", "feed price", "fish market price ", and "cost of vaccine dose". In the 20% cumulative mortality scenario, the net return would break-even (benefits ≥ costs) in at least 97.9% of interactions. Should cumulative mortality be lower than 10%, the profitability of vaccination would be more dependent on better feed conversion ratio. The inputs "feed price" and "cost of vaccine" had minor effects on the output, in all pre-vaccination mortality scenarios. Although our simulations are based on conservative values and consider uncertainty about the modeled parameters, we conclude that vaccination against S. agalactiae is likely to be profitable in Nile tilapia farms, under similar production conditions.

Design, statistical analysis and reporting standards for test accuracy studies for infectious diseases in animals: Progress, challenges and recommendations.

The quality of diagnostic accuracy studies (DAS) for infectious diseases of animals has improved over the last 20 years because of international educational efforts, use of design and reporting standards to guide researchers and test developers, and acceptance of the use of latent class models to account for imperfect reference tests. In this review, we focus on measurement of diagnostic sensitivity and specificity as a measure of clinical validity, describe the leadership role of the World Organisation of Animal Health (OIE) in setting standards for test validation in the context of fitness-for-purpose, and describe how design and reporting quality have facilitated the increased use of systematic reviews and meta-analysis of DAS. Ongoing challenges for design, conduct, analysis and reporting of DAS are identified; and we make recommendations for improvements in these areas for OIE-listed and non-listed infectious diseases.

Bayesian latent class analysis of ELISA and RT-rPCR diagnostic accuracy for subclinical Renibacterium salmoninarum infection in Atlantic salmon (Salmo salar) broodstock.

Renibacterium salmoninarum infection causes bacterial kidney disease (BKD) in salmonid freshwater and saltwater life stages, with potentially severe financial loss for the aquaculture industry. Preventing vertical transmission, from infected broodstock to eggs, is key to disease management. As there is no perfect reference standard for detecting R. salmoninarum, we used Bayesian latent class analyses to compare real-time reverse transcriptase PCR (RT-rPCR, mRNA target) and enzyme-linked immunosorbent assay (ELISA; p57 antigen target) diagnostic accuracy for detection in Atlantic salmon broodstock from British Columbia, Canada, and assessed ELISA repeatability. In 2016, 4,544 Atlantic salmon broodstock (no clinical signs of BKD or gross lesions) were sampled for ELISA testing of kidney tissue. Two groups of ELISA positives (n = 132) and two groups of a random sample of ELISA negatives (n = 137) were then tested with RT-rPCR, and ELISA testing was repeated. ELISA testing of broodstock provided the best diagnostic sensitivity (DSe; less chance of false-negative results). The use of joint RT-rPCR and ELISA testing improved DSe over that from each test alone, if a sample was considered positive when either test result was positive. Using these testing schemes in combination with management practices can decrease the likelihood of vertical transmission from subclinically infected broodstock.

Development and validation of an immunoperoxidase antigen detection test for improved diagnosis of rabies in Indonesia.

Rabies continues to pose a significant threat to human and animal health in regions of Indonesia. Indonesia has an extensive network of veterinary diagnostic laboratories and the 8 National laboratories are equipped to undertake diagnostic testing for rabies using the commercially-procured direct fluorescent antibody test (FAT), which is considered the reference (gold standard) test. However, many of the Indonesian Provincial diagnostic laboratories do not have a fluorescence microscope required to undertake the FAT. Instead, certain Provincial laboratories continue to screen samples using a chemical stain-based test (Seller's stain test, SST). This test has low diagnostic sensitivity, with negative SST-tested samples being forwarded to the nearest National laboratory resulting in significant delays for completion of testing and considerable additional costs. This study sought to develop a cost-effective and diagnostically-accurate immunoperoxidase antigen detection (RIAD) test for rabies that can be readily and quickly performed by the resource-constrained Provincial laboratories. This would reduce the burden on the National laboratories and allow more rapid diagnoses and implementation of post-exposure prophylaxis. The RIAD test was evaluated using brain smears fixed with acetone or formalin and its performance was validated by comparison with established rabies diagnostic tests used in Indonesia, including the SST and FAT. A proficiency testing panel was distributed between Provincial laboratories to assess the reproducibility of the test. The performance of the RIAD test was improved by using acetone fixation of brain smears rather than formalin fixation such that it was of equivalent accuracy to that of the World Organisation for Animal Health (OIE)-recommended FAT, with both tests returning median diagnostic sensitivity and specificity values of 0.989 and 0.993, respectively. The RIAD test and FAT had higher diagnostic sensitivity than the SST (median = 0.562). Proficiency testing using a panel of 6 coded samples distributed to 16 laboratories showed that the RIAD test had good reproducibility with an overall agreement of 97%. This study describes the successful development, characterisation and use of a novel RIAD test and its fitness for purpose as a screening test for use in provincial Indonesian veterinary laboratories.

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.

STARD-BLCM: Standards for the Reporting of Diagnostic accuracy studies that use Bayesian Latent Class Models.

The Standards for the Reporting of Diagnostic Accuracy (STARD) statement, which was recently updated to the STARD2015 statement, was developed to encourage complete and transparent reporting of test accuracy studies. Although STARD principles apply broadly, the checklist is limited to studies designed to evaluate the accuracy of tests when the disease status is determined from a perfect reference procedure or an imperfect one with known measures of test accuracy. However, a reference standard does not always exist, especially in the case of infectious diseases with a long latent period. In such cases, a valid alternative to classical test evaluation involves the use of latent class models that do not require a priori knowledge of disease status. Latent class models have been successfully implemented in a Bayesian framework for over 20 years. The objective of this work was to identify the STARD items that require modification and develop a modified version of STARD for studies that use Bayesian latent class analysis to estimate diagnostic test accuracy in the absence of a reference standard. Examples and elaborations for each of the modified items are provided. The new guidelines, termed STARD-BLCM (Standards for Reporting of Diagnostic accuracy studies that use Bayesian Latent Class Models), will facilitate improved quality of reporting on the design, conduct and results of diagnostic accuracy studies that use Bayesian latent class models.