Evaluation of histopathology, PCR, and qPCR to detect Mikrocytos mackini in oysters Crassostrea gigas using Bayesian latent class analysis.

Latent class analysis (LCA) is a common method to evaluate the diagnostic sensitivity (DSe) and specificity (DSp) for pathogen detection assays in the absence of a perfect reference standard. Here we used LCA to evaluate the diagnostic accuracy of 3 tests for the detection of Mikrocytos mackini in Pacific oysters Crassostrea gigas: conventional polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), and histopathology. A total of 802 Pacific oysters collected over 12 sampling events from 9 locations were assessed. Preliminary investigations indicated that standard LCA assumptions of test independence and constant detection accuracy across locations were likely unrealistic. This was mitigated by restructuring the LCA in a Bayesian framework to include test-derived knowledge about pathogen prevalence and load for categorizing populations into 2 classes of infection severity (low or high) and assessing separate DSe and DSp estimates for each class. Median DSp estimates were high (>96%) for all 3 tests in both population classes. DSe estimates varied between tests and population classes but were consistently highest for qPCR (87-99%) and lowest for histopathology (21-51%). Acknowledging that detection of M. mackini may be fitted to multiple diagnostic and management purposes, qPCR had the highest DSe while maintaining similar DSp to both conventional PCR and histopathology and thus is generally well-suited to most applications.

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.

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.

Infectious agent detections in archived Sockeye salmon (Oncorhynchus nerka) samples from British Columbia, Canada (1985-94).

In response to concerns that novel infectious agents were introduced through the movement of eggs as Atlantic salmon aquaculture developed in British Columbia (BC), Canada, we estimated the prevalence of infectious agents in archived return-migrating Sockeye salmon, from before and during aquaculture expansion in BC (1985-94). Of 45 infectious agents assessed through molecular assays in 652 samples, 23 (7 bacterial, 2 viral and 14 parasitic) were detected in liver tissue from six regions in BC. Prevalence ranged from 0.005 to 0.83 and varied significantly by region and year. Agent diversity ranged from 0 to 12 per fish (median 4), with the lowest diversity observed in fish from the Trans-Boundary and Central Coast regions. Agents known to be endemic in Sockeye salmon in BC, including Flavobacterium psychrophilum, Infectious haematopoietic necrosis virus, Ceratonova shasta and Parvicapsula minibicornis, were commonly observed. Others, such as Kudoa thyrsites and Piscirikettsia salmonis, were also detected. Surprisingly, infectious agents described only recently in BC salmon, Ca. Branchiomonas cysticola, Parvicapsula pseudobranchicola and Paranucleospora theridion, were also detected, indicating their potential presence prior to the expansion of the aquaculture industry. In general, our data suggest that agent distributions may not have substantially changed because of the salmon aquaculture industry.

In vivo screening of multiple bacterial strains identifies Lactobacillus rhamnosus Lb102 and Bifidobacterium animalis ssp. lactis Bf141 as probiotics that improve metabolic disorders in a mouse model of obesity.

Given the growing evidence that gut dysfunction, including changes in gut microbiota composition, plays a critical role in the development of inflammation and metabolic diseases, the identification of novel probiotic bacteria with immunometabolic properties has recently attracted more attention. Herein, bacterial strains were first isolated from dairy products and human feces and then screened in vitro for their immunomodulatory activity. Five selected strains were further analyzed in vivo, using a mouse model of diet-induced obesity. C57BL/6 mice were fed a high-fat high-sucrose diet, in combination with 1 of 3 Lactobacillus strains (Lb38, L. plantarum; L79, L. paracasei/casei; Lb102, L. rhamnosus) or Bifidobacterium strains (Bf26, Bf141, 2 different strains of B. animalis ssp. lactis species) administered for 8 wk at 109 colony-forming units/d. Whereas 3 strains showed only modest (Lb38, Bf26) or no (L79) effects, Lb102 and Bf141 reduced diet-induced obesity, visceral fat accretion, and inflammation, concomitant with improvement of glucose tolerance and insulin sensitivity. Further analysis revealed that Lb102 and Bf141 enhanced intestinal integrity markers in association with selective changes in gut microbiota composition. We have thus identified 2 new potential probiotic bacterial strains with immunometabolic properties to alleviate obesity development and associated metabolic disturbances.-Le Barz, M., Daniel, N., Varin, T. V., Naimi, S., Demers-Mathieu, V., Pilon, G., Audy, J., Laurin, E., Roy, D., Urdaci, M. C., St-Gelais, D., Fliss, I, Marette, A. In vivo screening of multiple bacterial strains identifies Lactobacillus rhamnosus Lb102 and Bifidobacterium animalis ssp. lactis Bf141 as probiotics that improve metabolic disorders in a mouse model of obesity.

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.

Detection and Assessment of the Distribution of Infectious Agents in Juvenile Fraser River Sockeye Salmon, Canada, in 2012 and 2013.

Infectious diseases may contribute to declines in Fraser River Sockeye salmon (Oncorhynchus nerka) stocks, but a clear knowledge gap exists around which infectious agents and diseases are important. This study was conducted to: (1) determine the presence and prevalence of 46 infectious agents in juvenile Fraser River Sockeye salmon, and (2) evaluate spatial patterns in prevalence and burden over initial seaward migration, contrasting patterns between 2 years of average and poor productivity. In total, 2,006 out-migrating Sockeye salmon were collected from four regions along their migration trajectory in British Columbia, in 2012 and 2013. High-throughput microfluidics quantitative PCR was employed for simultaneous quantitation of 46 different infectious agents. Twenty-six agents were detected at least once, including nine with prevalence >5%. Candidatus Brachiomonas cysticola, Myxobolus arcticus, and Pacific salmon parvovirus were the most prevalent agents. Infectious agent diversity and burden increased consistently upon smolts entry into the ocean, but they did not substantially change afterwards. Notably, both freshwater- and saltwater-transmitted agents were more prevalent in 2013 than in 2012, leading to an overall higher infection burden in the first two sampling regions. A reduction in the prevalence of two agents, erythrocytic necrosis virus and Paraneuclospora theridion, was observed between regions 2 and 3, which was speculated to be associated with mortality during the 1st month at sea. The most prevalent infectious agents were all naturally occurring. In a small number of samples (0.9%), seven agents were only detected around and after salmon farming regions, including four important pathogens: piscine orthoreovirus, Piscirickettsia salmonis, Tenacibaculum maritimum, and Moritella viscosa. As the first synoptic survey of infectious agents in juvenile Sockeye salmon in British Columbia, this study provides the necessary baseline for further research on the most prevalent infectious agents and their potential pathogenicity, which may adversely affect the productivity of valuable Sockeye salmon stocks. In addition, our findings are informative to the decision makers involved in conservation programs.

Serology in Finfish for Diagnosis, Surveillance, and Research: A Systematic Review.

Historically, serological tests for finfish diseases have been underused when compared with their use in terrestrial animal health. For years the nonspecific immune response in fish was judged to make serology unreliable and inferior to the direct measurement of agent analytes. We conducted a systematic review of peer-reviewed publications that reported on the development, validation, or application of serological tests for finfish diseases. A total of 168 articles met the screening criteria; most of them were focused on salmonid pathogens (e.g., Aeromonas spp. and viral hemorrhagic septicemia virus). Before the 1980s, most publications reported the use of agglutination tests, but our review indicates that enzyme-linked immunosorbent assay (ELISA) has more recently become the dominant serological test. The main application of serological tests has been in the assessment of vaccine efficacy, with few applications for surveillance or demonstration of freedom from disease, despite the advantages of serological tests over direct detection at the population level. Nonlethal sampling, low cost, and postinfection persistence of antibodies make serological assays the test of choice in surveillance, especially of valuable broodstock. However, their adoption has been constrained by poor characterization and validation. The number of publications in our review reporting diagnostic sensitivity and specificity of serological tests in finfish was small (n = 7). Foreseeing a wider use of serological tests in the future for diagnostic end purposes, we offer recommendations for mitigating deficiencies in the development and evaluation of serological tests, including optimization, control of nonspecific reactions, informed cutoff points, diagnostic accuracy, and serological baseline studies. Achieving these goals will facilitate greater international recognition of serological testing in programs supporting aquatic animal health. Received March 21, 2016; accepted September 24, 2016.

Assessment of the relative sensitivity of milk ELISA for detection of Mycobacterium avium ssp. paratuberculosis infectious dairy cows.

Milk ELISA are commonly used for detection of Mycobacterium avium ssp. paratuberculosis (MAP) antibodies in dairy cows, due to low cost and quick processing for large numbers of samples. However, low sensitivity and variations from host and environmental factors can impede detection of MAP antibodies at early disease stages. The objectives of our study were to assess the sensitivity of milk ELISA in comparison with fecal tests and to evaluate how detectable antibody concentrations in milk vary with changes in fecal shedding of MAP, cow age, cow parity, days in milk, and time of year. To compare the sensitivity of a commercial milk ELISA with solid and broth fecal culture and with fecal real-time PCR, a longitudinal study was performed for the identification of MAP-infectious animals as determined by prior fecal testing for MAP shedding. In addition, associations between variation in milk MAP ELISA score and changes in fecal MAP shedding, host age, days in milk, and season were evaluated. Monthly milk and fecal samples were collected over 1 yr from 46 cows that were previously shedding MAP in their feces. Sensitivity of milk ELISA was 29.9% (95% CI: 24.8 to 35.1%), compared with 46.7% (40.7 to 52.7%) for fecal solid culture, 55.0% (49.3 to 60.7%) for fecal broth culture, and 78.4% (73.3 to 83.1%) for fecal direct real-time PCR. The effect of stage of lactation could not be separated from the effect of season, with increased milk ELISA scores at greater days in milk in winter. However, unpredictable monthly variations in results were observed among the 3 assays for individual cow testing, which highlights the importance of identifying patterns in pathogen and antibody detection over time in MAP-positive herds.

Novel Cell Preservation Technique to Extend Bovine In Vitro White Blood Cell Viability.

Although cell-mediated immunity based diagnostics can be integral assays for early detection of various diseases of dairy cows, processing of blood samples for these tests is time-sensitive, often within 24 hours of collection, to maintain white blood cell viability. Therefore, to improve utility and practicality of such assays, the objective of this study was to assess the use of a novel white blood cell preservation technology in whole bovine blood. Blood samples from ten healthy cows were each divided into an unpreserved control sample and a test sample preserved with commercially-available cell transport medium. Samples were maintained at room temperature and stimulated with the mitogens pokeweed and concanavalinA, as well as with interleukin-12 p40. Stimulation was completed on days 1, 5, and 8 post-sampling. Viability of white blood cells was assessed through interferon gamma production determined with a commercial enzyme linked immunosorbent assay. In addition, mononuclear cell viability was assessed with propidium iodide flow cytometry. Greater interferon gamma production was observed on days 5 and 8 post-collection in preserved samples, with both pokeweed and concanavalinA stimulating positive interferon gamma production on day 5 post-collection. A greater proportion of the amount of interferon gamma produced on day 1 continued to be produced on days 5 and 8 post-collection with concanavalinA stimulation (with or without interleukin 12) as compared to pokeweed stimulation. Additionally, viable mononuclear cells were still present at eight days post-collection, with a higher mean proportion detected at days 5 and 8 in all stimulated preserved samples. This practical and simple method to extend in vitro white blood cell viability could benefit the efficient utilization of cell-based blood tests in ruminants.

Sensitivity of solid culture, broth culture, and real-time PCR assays for milk and colostrum samples from Mycobacterium avium ssp. paratuberculosis-infectious dairy cows.

Mycobacterium avium ssp. paratuberculosis (MAP) can be shed in feces, milk, and colostrum. The goal of this study was to assess assays that detect MAP in these sample types, including effects of lactation stage or season. Understanding the performance of these assays could improve how they are used, limiting the risk of infection to calves. Forty-six previously confirmed MAP-positive cows from 7 Atlantic Canadian dairy farms were identified for colostrum sampling and monthly sampling of milk and feces over a 12-mo period. Samples were assayed for MAP using solid culture, broth culture, and direct real-time PCR (qPCR). Across assay types, test sensitivity when applied to milk samples averaged 25% of that when applied to fecal samples. For colostrum samples, sensitivity depended on assay type, with sensitivity of qPCR being approximately 46% of that in feces. Across sample types, sensitivity of qPCR was higher than that of the other assays. Sensitivity of qPCR, when applied to milk samples, was significantly higher in summer than in other seasons. Summer was also the season with highest agreement between milk and fecal samples collected within the same month. Our results suggest that qPCR would detect more cows shedding MAP in their milk and colostrum than solid or broth culture assays, particularly during the summer, thus providing better management information to limit exposure of calves to this infectious organism.

The association of detection method, season, and lactation stage on identification of fecal shedding in Mycobacterium avium ssp. paratuberculosis infectious dairy cows.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative organism of Johne's disease. Although fecal culture is considered the standard diagnostic test, the long incubation times, costs, and intermittent shedding of MAP hinder efficient screening programs based on culture results. The primary objectives of this study were to determine the detection ability of solid culture, broth culture, and real-time PCR (qPCR) for MAP in fecal samples and to assess how shedding patterns of MAP in feces vary with lactation stage and season. This knowledge could improve the use of these diagnostic assays in Johne's management programs. For this study, 51 MAP-infectious cows from 7 Atlantic Canadian dairy farms had fecal samples collected monthly over a 12-mo period. Samples were analyzed for MAP bacterial load via solid culture, broth culture, and qPCR. For all fecal samples, 46% [95% confidence interval (CI): 40 to 51%] were positive by solid culture, 55% (95% CI: 50 to 60%) by broth culture, and 78% (95% CI: 73 to 82%) by qPCR. Sensitivity of qPCR was numerically higher in the dry and postpartum lactation periods, and qPCR detection in summer and fall was 85% of that in winter and spring. Furthermore, culture-determined moderate or light shedding categories generally corresponded to qPCR cycle threshold values <35, but heavy shedding categories corresponded to qPCR values <29. Direct fecal qPCR is a MAP detection method that is quick and less costly than culture techniques, and it avoids the use of decontamination steps that could decrease numbers of bacteria in a sample below the detection limit. This study indicates that, for known MAP-positive cows, fecal qPCR had high sensitivity of MAP detection, thereby supporting the use of direct fecal qPCR as part of a Johne's herd control program.

Impact of Bifidobacterium animalis subsp. lactis BB-12 and, Lactobacillus acidophilus LA-5-containing yoghurt, on fecal bacterial counts of healthy adults.

This randomized, placebo-controlled, double blind, parallel dose-response study investigated the impact of 4-week commercial yoghurt consumption supplemented with Bifidobacterium animalis subsp. lactis (BB-12) and Lactobacillus acidophilus (LA-5) on fecal bacterial counts of healthy adults. Fifty-eight volunteers were randomly assigned to three different groups: 1. placebo (no probiotic, no starter and no green tea extract); 2. Yoptimal (10(9)cfu/100g of BB-12 and LA-5 and 40mg of green tea extract) and 3. Yoptimal-10 (10(10)cfu/100g of BB-12, 10(9)cfu/100g of LA-5 and 40mg of green tea extract). These yoghurt products also contained Lactobacillus delbrueckii subsp. bulgaricus (10(7)cfu/100g) and Streptococcus thermophilus (10(10)cfu/100g). The quantitative PCR (qPCR) results showed that there were significant increases (P=0.02) in bifidobacteria counts with the Yoptimal treatment as compared to baseline. The fecal numbers of B. animalis subsp. lactis and LA-5 significantly increased in the two probiotic treatments compared to the placebo treatment. Viable counts of fecal lactobacilli were significantly higher (P=0.05) and those of enterococci were significantly lower (P=0.04) after the intervention when compared to placebo. No significant difference was observed between treatments in volunteers' weight, waist girth, blood pressure, fasting plasma triglyceride and HDL-C concentrations, as well as cholesterol/HDL-cholesterol ratio. However, a significant increase in plasma cholesterol levels was observed in the placebo group (P=0.0018) but the levels remained stable in the two probiotic yoghurt groups. These results show that probiotic strains supplemented in the form of yoghurt remain active during gut transit and are associated with an increase in beneficial bacteria and a reduction in potentially pathogenic bacteria. This trial was registered at clinicaltrials.gov as NCT00730626.