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.

Vertical transmission of Renibacterium salmoninarum in cutthroat trout (Oncorhynchus clarkii).

Vertical transmission of Renibacterium salmoninarum has been well-documented in anadromous salmonids but not in hatchery-reared inland trout. We assessed whether the bacterium is vertically transmitted in cutthroat trout (Oncorhynchus clarkii) from a Colorado, USA hatchery, and assessed the rate of transmission from male and female brood fish. Adult brood fish were killed, tested for R. salmoninarum in kidney, liver, spleen, ovarian fluid, blood and mucus samples, then stripped of gametes to create 32 families with four infection treatments (MNFN, MNFP, MPFN, MPFP; M: male, F: female, P: positive, N: negative). Progeny from each treatment was sampled at 6 and 12 months to test for the presence of R. salmoninarum with an enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Our study indicated that vertical transmission was high and occurred among 60% of families across all infection treatments. However, the average proportion of infected progeny from individual families was low, ranging from 1% (MNFP, MPFN and MPFP treatments) up to 21% (MPFP treatment). Hatcheries rearing inland salmonids would be well suited to limit vertical transmission through practices such as lethal culling because any amount of transmission can perpetuate the infection throughout fish on a hatchery.

Method for lineage typing of epidemic Renibacterium salmoninarum in Chilean salmon farms.

Renibacterium salmoninarum (Rs) is the etiological agent of bacterial kidney disease (BKD), which significantly affects farmed and wild salmonids worldwide. Although the whole genome of Rs (~3.1 million nucleotides) is highly conserved, genomic epidemiology analyses have identified four sub-lineages from Chilean isolates. A total of 94 Rs genomes from the BIGSdb aquaculture database were aligned and compared using bioinformatics tools, identifying 2199 independent single-nucleotide polymorphisms (SNPs) spread along the genome. A detailed analysis of the distribution of the SNPs showed five local zones of a length in the range of 10-15 kbp that should be used to unambiguously identify a specific sub-lineage. Based on the Rs type strain DSM 20767T , we designed multiplex PCR primers that produce specific amplification products which were further sequenced by the Sanger method to obtain the genotype of the sub-lineage. For the genetic typing, we evaluated 27 Rs isolates recovered from BKD outbreaks from different fish species and regions of Chile. Based on the findings reported here, we propose the PCR approach as a valuable tool for the rapid and reliable studying of the relationships between Rs isolates and the different sub-lineages without requiring the sequencing of the entire genome.

Comparison of injection and immersion challenges of Renibacterium salmoninarum strains in Rainbow Trout.

OBJECTIVE: Renibacterium salmoninarum is a pathogenic gram-positive bacterium and is the causative agent of bacterial kidney disease (BKD), a malady that mainly impacts salmonid species. Experimental challenges were conducted to assess the virulence and challenge route for select R. salmoninarum strains (CK-90 and ATCC 33739) in Rainbow Trout Oncorhynchus mykiss. METHODS: The CK-90 strain was intracoelomically injected (100 µL) at a high dose containing 4.80 × 106 CFU/g of fish (optical density at 525 nm [OD525 ] = 1.779) and a low dose containing 6.86 × 105 CFU/g of fish (OD525  = 1.077); alternatively, fish were immersed in a solution containing 4.5 × 107 CFU/mL of fish (OD525  = 0.886). The ATCC 33739 strain (originating from Brook Trout Salvelinus fontinalis) was also included and intracoelomically injected at 3.58 × 105 CFU/g of fish (OD525  = 1.431) to discern differences in virulence between the strains. RESULT: Clinical signs of BKD manifested at approximately 10 d postchallenge, and mortalities began at 19 days postchallenge. To confirm infection and quantify R. salmoninarum antigen load, an enzyme-linked immunosorbent assay (ELISA) was conducted using kidney tissue collected after the challenge. Rainbow Trout that were challenged with CK-90 by injection (both high- and low-dose groups) exhibited significantly higher mortality than fish that were injected with ATCC 33739 or those that were exposed to CK-90 via immersion challenge. The R. salmoninarum p57 (57-kDa protein) antigen was confirmed via ELISA. Antigen load for fish injected with CK-90 (high dose: OD405  = 0.71; low dose: OD405  = 0.66) was significantly higher than that for fish injected with ATCC 33739 (OD405  = 0.34). The CK-90 strain (both high and low doses) was more virulent than ATCC 33739, which caused no mortalities over the 28-days trial. Although there were no mortalities among ATCC 33739 fish, the ELISA confirmed that the R. salmoninarum antigen infiltrated kidney tissue in those fish. CONCLUSION: The immersion challenge methodology for R. salmoninarum CK-90 was ineffective for inducing mortalities at the examined dose.

Prevalence and distribution of Renibacterium salmoninarum, causative agent of bacterial kidney disease, in wild trout fisheries in Colorado.

Detections of Renibacterium salmoninarum in Colorado USA fish hatcheries have become more frequent in recent years, including one disease outbreak that originated with a wild broodstock. Our objectives were to document the prevalence and distribution of R. salmoninarum in Colorado's wild trout fisheries, investigate variables that influence that distribution, and evaluate the effectiveness of common testing methods on non-anadromous trout. We sampled wild trout across Colorado and tested kidney tissue with enzyme-linked immunosorbent assay (ELISA), quantitative polymerase chain reaction (qPCR), nested polymerase chain reaction (nPCR), and direct fluorescent antibody test (DFAT). Screening with ELISA showed high prevalence of R. salmoninarum among fish populations, but antigen levels were low. No clinical disease was observed in any of the fish sampled despite the antigen of R. salmoninarum being common. Antigen levels measured by ELISA increased in smaller streams with lower historic fish stocking rates. Brook trout Salvelinus fontinalis had the highest prevalence of the bacterium among fish species and highest ELISA antigen levels. The distribution of brook trout in the smallest streams may help explain the patterns of R. salmoninarum across the landscape. The most effective assays for screening wild trout were qPCR and ELISA; DFAT was inconsistent for bacterial levels encountered in wild trout and generally uninformative. Additionally, qPCR and ELISA can provide quantitative information about bacteria levels. The bacterium R. salmoninarum is ubiquitous in Colorado trout fisheries but is generally found at low levels. Active infections are rare and overt bacterial kidney disease appears more common in Colorado hatcheries than in wild fish.

Proteomic analysis reveals Renibacterium salmoninarum grown under iron-limited conditions induces iron uptake mechanisms and overproduction of the 57-kDa protein.

Renibacterium salmoninarum, a slow-growing facultative intracellular pathogen, is the causative agent of bacterial kidney disease, a chronic, progressive and granulomatous infection that threatens farmed and wild salmonids worldwide. Pathogenic R. salmoninarum colonizes tissues and invades the host through cell surface-associated and secreted proteins. While correlations between iron acquisition genes and virulence have been demonstrated in vitro, these mechanisms have not undergone proteomic characterization. The present study applied a proteomic approach to elucidate the differences between the virulent Chilean R. salmoninarum H-2 strain and the type strain ATCC 33209T . Analyses were conducted under normal (control) and iron-limited conditions (DIP) emulating the host environment. Interestingly, strain H-2 apparently responded better to the iron-limited condition-for example, only this strain presented a significantly enriched iron ion homeostasis pathway. Furthermore, key virulence factors related to an iron-limited environment were more abundant in strain H-2. Importantly, the lack of iron favoured the expression of the 57-kDa protein in strain H-2, the principal virulence factor for R. salmoninarum. Our findings can be employed in the design and development of treatments targeted to iron uptake mechanisms (e.g. siderophore synthesis or haem uptake), which represents a promising therapeutic approach for treating this persistent fastidious bacterium.

Isolation and Characterization of a ssDNA Aptamer against Major Soluble Antigen of Renibacterium salmoninarum.

Bacterial kidney disease (BKD) is a major health problem of salmonids, affecting both wild and cultured salmon. The disease is caused by Renibacterium salmoninarum (Rs), a fastidious, slow-growing and strongly Gram-positive diplobacillus that produces chronic, systemic infection characterized by granulomatous lesions in the kidney and other organs, often resulting in death. Fast detection of the pathogen is important to limit the spread of the disease, particularly in hatcheries or aquaculture facilities. Aptamers are increasingly replacing conventional antibodies as platforms for the development of rapid diagnostic tools. In this work, we describe the first instance of isolating and characterizing a ssDNA aptamer that binds with high affinity to p57 or major soluble antigen (MSA), the principal antigen found on the cell wall surface of Rs. Specifically, in this study a construct of the full-length protein containing a DNA binding domain (MSA-R2c) was utilized as target. Aptamers were isolated from a pool of random sequences using GO-SELEX (graphene oxide-systematic evolution of ligands by exponential enrichment) protocol. The selection generated multiple aptamers with conserved motifs in the random region. One aptamer with high frequency of occurrence in different clones was characterized and found to display a strong binding affinity to MSA-R2c with a Kd of 3.0 ± 0.6 nM. The aptamer could be potentially utilized for the future development of a sensor for rapid and onsite detection of Rs in water or in infected salmonids, replacing time-consuming and costly lab analyses.

Profiling the transcriptome response of Atlantic salmon head kidney to formalin-killed Renibacterium salmoninarum.

Renibacterium salmoninarum is a Gram-positive, intracellular bacterial pathogen that causes Bacterial Kidney Disease (BKD) in Atlantic salmon (Salmo salar). The host transcriptomic response to this immune-suppressive pathogen remains poorly understood. To identify R. salmoninarum-responsive genes, Atlantic salmon were intraperitoneally injected with a low (5 × 105 cells/kg, Low-Rs) or high (5 × 107 cells/kg; High-Rs) dose of formalin-killed R. salmoninarum bacterin or phosphate-buffered saline (PBS control); head kidney samples were collected before and 24 h after injection. Using 44K microarray analysis, we identified 107 and 345 differentially expressed probes in response to R. salmoninarum bacterin (i.e. High-Rs vs. PBS control) by Significance Analysis of Microarrays (SAM) and Rank Products (RP), respectively. Twenty-two microarray-identified genes were subjected to qPCR assays, and 17 genes were confirmed as being significantly responsive to the bacterin. There was an up-regulation in expression of genes playing putative roles as immune receptors and antimicrobial effectors. Genes with putative roles as pathogen recognition (e.g. clec12b and tlr5) or immunoregulatory (e.g. tnfrsf6b and tnfrsf11b) receptors were up-regulated in response to R.salmoninarum bacterin. Also, chemokines and a chemokine receptor showed opposite regulation [up-regulation of effectors (i.e. ccl13 and ccl) and down-regulation of cxcr1] in response to the bacterin. The present study identified and validated novel biomarker genes (e.g. ctsl1, lipe, cldn4, ccny) that can be used to assess Atlantic salmon response to R. salmoninarum, and will be valuable in the development of tools to combat BKD.

First report of bacterial kidney disease in coho salmon Oncorhynchus kisutch in Russia.

This paper describes the first case of bacterial kidney disease (BKD) to be identified in coho salmon Oncorhynchus kisutch in Russia. The fish in question was caught in Lake Bolshoi Vilyui on the Kamchatka Peninsula. The diseased fish had foci of granulomatous inflammation in the kidneys. The diagnosis was confirmed by isolating the bacterium Renibacterium salmoninarum from kidney tissue in pure culture, and by determining the partial 16S RNA gene sequence of the isolate. This is the first detection of this pathogen in the genus Oncorhynchus in Russia, and detection of BKD in coho salmon indicates that the pathogen is present in the natural fish populations of Kamchatka. Therefore, it will be necessary to conduct screening studies of mature salmon selected for artificial reproduction, for the presence of BKD signs and asymptomatic infection with R. salmoninarum, which will allow us to estimate the prevalence of the pathogen.

Variations of the intestinal gut microbiota of farmed rainbow trout, Oncorhynchus mykiss (Walbaum), depending on the infection status of the fish.

AIMS: The aim of the present study was to investigate the composition of the intestinal microbiota during the acute stage of a bacterial infection to understand how dysbiosis of the gut may influence overall taxonomic hierarchy and diversity, and determine if there exists a bacterial taxon(s) that serve as markers for healthy or diseased rainbow trout (Oncorhynchus mykiss). METHODS AND RESULTS: From July to September 2015, 29 specimens of 3-year-old (an average weight from 240·9 ± 37·7 to 850·7 ± 70·1 g) rainbow trout O. mykiss were studied. Next-generation high-throughput sequencing of the 16S ribosomal RNA genes was applied to stomach and intestinal samples to compare the impact of infection status on the microbiota of rainbow trout O. mykiss (Walbaum) from the northwest part of Eurasia (Karelian region, Russia). The alpha diversity (Chao1, Simpson and Shannon index) of the microbial community of healthy rainbow trout was significantly higher than in unhealthy fish. The greatest contribution to the gut microbial composition of healthy fish was made by OTU's belonging to Bacillus, Serratia, Pseudomonas, Cetobacterium and Lactobacillus. Microbiota of unhealthy fish in most cases was represented by the genera Serratia, Bacillus and Pseudomonas. In microbiota of unhealthy fish there were also registered unique taxa such as bacteria from the family Mycoplasmataceae and Renibacterium. Analysis of similarities test revealed the significant dissimilarity between the microbiota of stomach and intestine (P ≤ 0·05). CONCLUSIONS: A substantial finding was the absence of differences between microbial communities of the stomach and intestine in the unhealthy groups if compared with healthy fish. SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrated alterations of the gut microbiota of farmed rainbow trout, O. mykiss during co-infections and can be useful for the development of new strategies for disease control programs.

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.

Case definition for clinical and subclinical bacterial kidney disease (BKD) in Atlantic Salmon (Salmo salar L.) in New Brunswick, Canada.

Bacterial kidney disease (BKD) is considered an important cause of loss in salmon aquaculture in Atlantic Canada. Causative agent of BKD is the Gram-positive bacteria Renibacterium salmoninarum. Infected salmon are often asymptomatic (subclinical infection), and the disease is considered chronic. One of the challenges in quantifying information from farm production and health records is the application of a standardized case definition. Case definitions for farm-level and cage-level clinical and subclinical BKD were developed using retrospective longitudinal data from aquaculture practices in New Brunswick, Canada, combining (i) industry records of weekly production data including mortalities, (ii) field observations for BKD using reports of veterinarians and/or fish health technicians, (iii) diagnostic submissions and test results and (iv) treatments used to control BKD. Case definitions were evaluated using veterinarians' expert judgements as reference standard. Eighty-nine and 66% of sites and fish groups, respectively, were associated with BKD at least once. For BKD present (subclinical or clinical), sensitivity and specificity of the case definition were 75-100% varying between event, fish group, site cycle and level (site pen). For clinical BKD, sensitivities were 29-64% and specificities 91-100%. Industry data can be used to develop sensitive case definitions.

Levels of Renibacterium salmoninarum antigens in resident and anadromous salmonids in the River Ellidaár system in Iceland.

In relation to stock enhancement programmes, wild salmon broodfish have been routinely screened for the presence of Renibacterium salmoninarum antigens (Rs-Ag) for decades. A sudden increase in the prevalence of Rs-Ag experienced caused extensive problems to this industry as eggs from positive fish are discarded. The prevalence and level of Rs-Ag were examined in resident and anadromous salmonids in the River Ellidaár system and the progress of Rs-Ag in a cohort of salmon followed. Both prevalence and Rs-Ag levels were high in resident salmonids and emigrating salmon smolts in the river system. When the smolts re-entered their home river as adults the following summer, they were almost free of Rs-Ag, but the longer they stayed in the river, the more Rs-Ag they acquired; the majority being positive at spawning. This study demonstrates a high level of Rs-Ag in salmonids in the River Ellidaár system which significantly reduces in the salmon during its seawater phase. Accordingly, it seems ideal to sample salmon broodfish as soon as possible after ascending the river and subsequently transfer to Rs-free environment for storage until stripping, which could result in lower Rs-prevalence and minimize the problems that stock enhancement programmes have faced due to Rs-positive wild broodfish.

Iron acquisition and siderophore production in the fish pathogen Renibacterium salmoninarum.

Renibacterium salmoninarum is the causative agent of bacterial kidney disease, which significantly affects salmonid farming worldwide. Despite this impact, there is scarce data on its iron uptake ability, a factor of pathogenesis. This study investigated the iron acquisition mechanisms of R. salmoninarum and its capacity to uptake iron from different sources. Thirty-two Chilean isolates and the DSM20767T type strain grew in the presence of 2,2'-Dipyridyl at varying concentrations (250-330 µm), and all isolates positively reacted on chrome azurol S agar. Subsequently, inocula of four Chilean isolates and the type strain were prepared with or without 200 µm of 2,2'-Dipyridyl for uptake assays. Assay results revealed differences between the isolates in terms of iron acquisition. While a prior iron-limited environment was, for most isolates, not required to activate the uptake of iron (II) sulphate, ammonium iron (III) citrate or iron (III) chloride at higher concentrations (100 µm), it did facilitate growth at lower iron concentrations (10 µm and 1 µm). An exception was the H-2 isolate, which only grew with 100 µm of iron sulphide. In turn, 100 µm of haemin was toxic when isolates were grown in normal KDM-2. In silico R. salmoninarumATCC 33209T genome analysis detected various genes coding iron uptake-related proteins. This is the first study indicating two iron acquisition systems in R. salmoninarum: one involving siderophores and another involving haem group utilization. These data represent a first step towards fully elucidating this virulence factor in the pathogenic R. salmoninarum.

Transcriptome profiling of lumpfish (Cyclopterus lumpus) head kidney to Renibacterium salmoninarum at early and chronic infection stages.

Renibacterium salmoninarum causes Bacterial Kidney Disease (BKD) in several fish species. Atlantic lumpfish, a cleaner fish, is susceptible to R. salmoninarum. To profile the transcriptome response of lumpfish to R. salmoninarum at early and chronic infection stages, fish were intraperitoneally injected with either a high dose of R. salmoninarum (1 × 109 cells dose-1) or PBS (control). Head kidney tissue samples were collected at 28- and 98-days post-infection (dpi) for RNA sequencing. Transcriptomic profiling identified 1971 and 139 differentially expressed genes (DEGs) in infected compared with control samples at 28 and 98 dpi, respectively. At 28 dpi, R. salmoninarum-induced genes (n = 434) mainly involved in innate and adaptive immune response-related pathways, whereas R. salmoninarum-suppressed genes (n = 1537) were largely connected to amino acid metabolism and cellular processes. Cell-mediated immunity-related genes showed dysregulation at 98 dpi. Several immune-signalling pathways were dysregulated in response to R. salmoninarum, including apoptosis, alternative complement, JAK-STAT signalling, and MHC-I dependent pathways. In summary, R. salmoninarum causes immune suppression at early infection, whereas lumpfish induce a cell-mediated immune response at chronic infection. This study provides a complete depiction of diverse immune mechanisms dysregulated by R. salmoninarum in lumpfish and opens new avenues to develop immune prophylactic tools to prevent BKD.

ISOTHERMAL RECOMBINANT POLYMERASE AMPLIFICATION AND CRIPSR(CAS12A) ASSAY DETECTION OF RENIBACTERIUM SALMONINARUM AS AN EXAMPLE FOR WILDLIFE PATHOGEN DETECTION IN ENVIRONMENTAL DNA SAMPLES.

Improving rapid detection methods for pathogens is important for research as we collectively aim to improve the health of ecosystems globally. In the northern hemisphere, the success of salmon (Oncorhynchus spp.) populations is vitally important to the larger marine, aquatic, and terrestrial ecosystems they inhabit. This has led to managers cultivating salmon in hatcheries and aquaculture to bolster their populations, but young salmon face many challenges, including diseases such as bacterial kidney disease (BKD). Early detection of the BKD causative agent, Renibacterium salmoninarum, is useful for managers to avoid outbreaks in hatcheries and aquaculture stocks to enable rapid treatment with targeted antibiotics. Isothermal amplification and CRIPSR-Cas12a systems may enable sensitive, relatively rapid, detection of target DNA molecules from environmental samples compared to quantitative PCR (qPCR) and culture methods. We used these technologies to develop a sensitive and specific rapid assay to detect R. salmoninarum from water samples using isothermal recombinase polymerase amplification (RPA) and an AsCas12a RNA-guided nuclease detection. The assay was specific to R. salmoninarum (0/10 co-occurring or closely related bacteria detected) and sensitive to 0.0128 pg/µL of DNA (approximately 20-40 copies/µL) within 10 min of Cas activity. This assay successfully detected R. salmoninarum environmental DNA in 14/20 water samples from hatcheries with known quantification for the pathogen via previous qPCR (70% of qPCR-positive samples). The RPA-CRISPR/AsCas12a assay had a limit of detection (LOD) of >10 copies/µL in the hatchery water samples and stochastic detection below 10 copies/µL, similar to but slightly higher than the qPCR assay. This LOD enables 37 C isothermal detection, potentially in the field, of biologically relevant levels of R. salmoninarum in water. Further research is needed to develop easy-to-use, cost-effective, sensitive RPA/CRISPR-AsCas12a assays for rapidly detecting low concentrations of wildlife pathogens in environmental samples.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) 2016/429): Bacterial kidney disease (BKD).

Bacterial kidney disease (BKD) was assessed according to the criteria of the Animal Health Law (AHL), in particular the criteria of Article 7 on disease profile and impacts, Article 5 on its eligibility to be listed, Annex IV for its categorisation according to disease prevention and control rules as laid out in Article 9 and Article 8 for listing animal species related to BKD. The assessment was performed following the ad hoc method on data collection and assessment developed by AHAW Panel and already published. The outcome reported is the median of the probability ranges provided by the experts, which indicates whether each criterion is fulfilled (lower bound ≥ 66%) or not (upper bound ≤ 33%), or whether there is uncertainty about fulfilment. Reasoning points are reported for criteria with an uncertain outcome. According to this assessment, BKD can be considered eligible to be listed for Union intervention according to Article 5 of the AHL (66-90% probability). According to the criteria in Annex IV, for the purpose of categorisation related to the level of prevention and control as in Article 9 of the AHL, the AHAW Panel concluded that BKD does not meet the criteria in Sections 1, 2 and 3 (Categories A, B and C; 1-5%, 33-66% and 33-66% probability of meeting the criteria, respectively) but meets the criteria in Sections 4 and 5 (Categories D and E; 66-90% and 66-90% probability of meeting the criteria, respectively). The animal species to be listed for BKD according to Article 8 criteria are provided.

Nutritional immunomodulation of Atlantic salmon response to Renibacterium salmoninarum bacterin.

We investigated the immunomodulatory effect of varying levels of dietary ω6/ω3 fatty acids (FA) on Atlantic salmon (Salmo salar) antibacterial response. Two groups were fed either high-18:3ω3 or high-18:2ω6 FA diets for 8 weeks, and a third group was fed for 4 weeks on the high-18:2ω6 diet followed by 4 weeks on the high-18:3ω3 diet and termed "switched-diet". Following the second 4 weeks of feeding (i.e., at 8 weeks), head kidney tissues from all groups were sampled for FA analysis. Fish were then intraperitoneally injected with either a formalin-killed Renibacterium salmoninarum bacterin (5 × 107 cells mL-1) or phosphate-buffered saline (PBS control), and head kidney tissues for gene expression analysis were sampled at 24 h post-injection. FA analysis showed that the head kidney profile reflected the dietary FA, especially for C18 FAs. The qPCR analyses of twenty-three genes showed that both the high-ω6 and high-ω3 groups had significant bacterin-dependent induction of some transcripts involved in lipid metabolism (ch25ha and lipe), pathogen recognition (clec12b and tlr5), and immune effectors (znrf1 and cish). In contrast, these transcripts did not significantly respond to the bacterin in the "switched-diet" group. Concurrently, biomarkers encoding proteins with putative roles in biotic inflammatory response (tnfrsf6b) and dendritic cell maturation (ccl13) were upregulated, and a chemokine receptor (cxcr1) was downregulated with the bacterin injection regardless of the experimental diets. On the other hand, an inflammatory regulator biomarker, bcl3, was only significantly upregulated in the high-ω3 fed group, and a C-type lectin family member (clec3a) was only significantly downregulated in the switched-diet group with the bacterin injection (compared with diet-matched PBS-injected controls). Transcript fold-change (FC: bacterin/PBS) showed that tlr5 was significantly over 2-fold higher in the high-18:2ω6 diet group compared with other diet groups. FC and FA associations highlighted the role of DGLA (20:3ω6; anti-inflammatory) and/or EPA (20:5ω3; anti-inflammatory) vs. ARA (20:4ω6; pro-inflammatory) as representative of the anti-inflammatory/pro-inflammatory balance between eicosanoid precursors. Also, the correlations revealed associations of FA proportions (% total FA) and FA ratios with several eicosanoid and immune receptor biomarkers (e.g., DGLA/ARA significant positive correlation with pgds, 5loxa, 5loxb, tlr5, and cxcr1). In summary, dietary FA profiles and/or regimens modulated the expression of some immune-relevant genes in Atlantic salmon injected with R. salmoninarum bacterin. The modulation of Atlantic salmon responses to bacterial pathogens and their associated antigens using high-ω6/high-ω3 diets warrants further investigation.

Evidence for the Use of Mucus Swabs to Detect Renibacterium salmoninarum in Brook Trout.

Efforts to advance fish health diagnostics have been highlighted in many studies to improve the detection of pathogens in aquaculture facilities and wild fish populations. Typically, the detection of a pathogen has required sacrificing fish; however, many hatcheries have valuable and sometimes irreplaceable broodstocks, and lethal sampling is undesirable. Therefore, the development of non-lethal detection methods is a high priority. The goal of our study was to compare non-lethal sampling methods with standardized lethal kidney tissue sampling that is used to detect Renibacterium salmoninarum infections in salmonids. We collected anal, buccal, and mucus swabs (non-lethal qPCR) and kidney tissue samples (lethal DFAT) from 72 adult brook trout (Salvelinus fontinalis) reared at the Colorado Parks and Wildlife Pitkin Brood Unit and tested each sample to assess R. salmoninarum infections. Standard kidney tissue detected R. salmoninarum 1.59 times more often than mucus swabs, compared to 10.43 and 13.16 times more often than buccal or anal swabs, respectively, indicating mucus swabs were the most effective and may be a useful non-lethal method. Our study highlights the potential of non-lethal mucus swabs to sample for R. salmoninarum and suggests future studies are needed to refine this technique for use in aquaculture facilities and wild populations of inland salmonids.

HIGH PREVALENCE OF CIRCULATING ANTIBODIES TO RENIBACTERIUM SALMONINARUM IN SPAWNING ONCORHYNCHUS SPP. FROM LAKE MICHIGAN, USA.

Bacterial kidney disease, caused by Renibacterium salmoninarum, threatens salmonids worldwide. Following devastating mortality episodes in Oncorhynchus spp. in Lake Michigan, US, in the 1980s and infection rates >90%, pathogen prevalence has steadily declined to <5% over three decades in the three state-managed stocks. In this study, we sought to determine if the declining infection rates were associated with heightened circulating antibodies in state-managed Oncorhynchus spp. residing in the Lake Michigan watershed. A single-dilution, indirect enzyme-linked immunosorbent assay (ELISA) was modified to detect circulating antibodies against R. salmoninarum. Baseline values were delineated from naive chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchus mykiss). The assay was first used to assess primary antibody production over a 4-wk period in chinook salmon experimentally infected with R. salmoninarum. Mean antibody response was detected as early as 2 wk postinfection and continued to increase to the end of the observation period. The modified ELISA was then used to detect antibodies in serum samples collected from feral adult chinook salmon, coho salmon (Oncorhynchus kisutch), and steelhead trout (O. mykiss) returning to spawn at Lake Michigan weirs in 2009 and 2013. Results demonstrated that about 80% of feral Oncorhynchus spp. had measurable titers of circulating antibodies to R. salmoninarum. The relative ease and reasonable costs of this modified ELISA makes it a valuable serosurveillance tool for assessing the humoral immune status of feral salmonid populations.