Development of RT-qPCR for quantification of human enterovirus D68 in vitro.

The common cold is the most frequent viral infectious disease of the upper respiratory tract with different intensities based on the serotype and the characteristics of the virus. Numerous human rhinoviruses have been identified and classified. Human rhinovirus 87 (HRV87), also known as enterovirus D68 (EV-D68), is one of the common viruses causing respiratory infections. In this study, a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay was developed, optimized, and validated for the detection of EV-D68. Method development also covers specificity, sensitivity, efficiency, and inter-and-intra-assay variations. Overall, this one-step qPCR assay will permit quantitative assessments of human enterovirus D68 RNA.•Enterovirus D68 is a reemerging viral agent causing respiratory infection.•RT-qPCR assay developed for detection of human enterovirus D68.•In this article validation to secure reproducibility is done according to MIQE guidelines.

Losing the 'arms race': multiresistant salmon lice are dispersed throughout the North Atlantic Ocean.

Nothing lasts forever, including the effect of chemicals aimed to control pests in food production. As old pesticides have been compromised by emerging resistance, new ones have been introduced and turned the odds back in our favour. With time, however, some pests have developed multi-pesticide resistance, challenging our ability to control them. In salmonid aquaculture, the ectoparasitic salmon louse has developed resistance to most of the available delousing compounds. The discovery of genetic markers associated with resistance to organophosphates and pyrethroids made it possible for us to investigate simultaneous resistance to both compounds in approximately 2000 samples of salmon lice from throughout the North Atlantic in the years 2000-2016. We observed widespread and increasing multiresistance on the European side of the Atlantic, particularly in areas with intensive aquaculture. Multiresistant lice were also found on wild Atlantic salmon and sea trout, and also on farmed salmonid hosts in areas where delousing chemicals have not been used. In areas with intensive aquaculture, there are almost no lice left that are sensitive to both compounds. These results demonstrate the speed to which this parasite can develop widespread multiresistance, illustrating why the aquaculture industry has repeatedly lost the arms race with this highly problematic parasite.

A salty start: Brackish water start-up as a microbial management strategy for nitrifying bioreactors with variable salinity.

Nitrifying biofilms developed in brackish water are reported to be more robust to salinity changes than freshwater biofilms. This makes them a promising strategy for water treatment systems with variable salinity, such as recirculating aquaculture systems for Atlantic salmon. However, little is known about the time required for nitrification start-up in brackish water or the microbial community dynamics. To investigate the development of nitrifying biofilms at intermediate salinity, we compared the startup of moving bed biofilm reactors with virgin carriers in brackish- (12‰ salinity) and freshwater. After 60 days, the brackish water biofilm had half the nitrification capacity of the freshwater biofilm, with a less diverse microbial community, lower proportion of nitrifiers, and a significantly different nitrifying community composition. Nitrosomonas and Nitrosospira-like bacteria were the main ammonia oxidizers in the brackish water biofilms, whereas Nitrosomonas was dominant in freshwater biofilms. Nitrotoga was the dominant nitrite oxidizer in both treatments. Despite the lower nitrification capacity in the brackish water treatment, the low ammonia and nitrite concentration with rapidly increasing nitrate concentration indicated that complete nitrification was established in both reactors within 60 days. The results suggest that biofilms develop nitrification in brackish water in comparable time as in freshwater, and brackish start-up can be a strategy for bioreactors with varying salinity.

Biofilms remember: Osmotic stress priming as a microbial management strategy for improving salinity acclimation in nitrifying biofilms.

With increasing freshwater scarcity and greater use of seawater, fluctuating salinities are becoming common in water treatment systems. This can be challenging for salinity-sensitive processes like nitrification, especially in recirculating aquaculture systems (RAS), where maintaining nitrification efficiency is crucial for fish health. This study was undertaken to determine if prior exposure to seawater (priming) could improve nitrification in moving bed biofilm reactors (MBBR) under salinity increase from freshwater to seawater. The results showed that seawater-primed freshwater MBBRs had less than 10% reduction in nitrification activity and twice the ammonia oxidation capacity of the unprimed bioreactors after seawater transfer. The primed biofilms had different microbial community composition but the same nitrifying taxa, suggesting that priming promoted physiological adaptation of the nitrifiers. Priming may also have strengthened the extrapolymeric matrix protecting the nitrifiers. In MBBRs started up in brackish water (12‰ salinity), seawater priming had no significant impact on the nitrification activity and the microbial community composition. These bioreactors were inherently robust to salinity increase, likely because they were already primed to osmotic stress by virtue of their native salinity of 12‰. The results show that osmotic stress priming is an effective strategy for improving salinity acclimation in nitrifying biofilms and can be applied to water treatment systems where salinity variations are expected.

The Phe362Tyr mutation conveying resistance to organophosphates occurs in high frequencies in salmon lice collected from wild salmon and trout.

The parasitic salmon louse, and its resistance to chemical delousing agents, represents one of the largest challenges to the salmon aquaculture industry. We genotyped lice sampled from wild salmon and sea trout throughout Norway with the recently identified Phe362Tyr mutation that conveys resistance to organophosphates. These results were compared to data from lice sampled on farmed salmon in the same regions. The resistant (R) allele was observed in salmon lice from wild salmon and sea trout throughout Norway, although its frequency was highest in farming-intense regions. In most regions, the frequency of the R allele was higher in lice collected from wild sea trout than wild Atlantic salmon, and in all regions, the frequency of the R allele was similar in lice collected from wild sea trout and farmed Atlantic salmon. The R allele is only selected for in fish-farms where organophosphates are used for delousing. Therefore, our results suggest extensive exchange of lice between farmed and wild hosts, and indicate that in farming-dense regions in Norway, aquaculture represents a major driver of salmon louse population structure. Finally, these data suggest that the wild hosts within the regions studied will not delay the spread of resistance when organophosphates are used.

Establishment of a non-invasive method for stress evaluation in farmed salmon based on direct fecal corticoid metabolites measurement.

Fish welfare is an important issue for growth of the aquaculture industry. Stress responses represent animal's natural reactions to challenging conditions and could be used as a welfare indicator. Cortisol level is relevant to fish welfare condition, and is a readily measured component of the primary stress response system. Generally, cortisol is measured by blood sampling. However, fish blood cortisol level could be instantly influenced by handling-stress at sampling. Fecal corticoid metabolites (FCM) are a mixture of several different metabolites with a wide range of polarities. Thus, feces could be promising alternative less handling-sensitive and non-invasive biological matrices for cortisol evaluation in Atlantic salmon. In this study we developed non-invasive method for determination of fecal corticoid metabolites in farmed Atlantic salmon (Salmo salar L.) using enzyme-linked immunosorbent assay (ELISA). It was demonstrated that salmon FCM extracted from salmon feces is insoluble in non-polar solvents like diethyl ether, but well soluble in polar solvents like methanol. The proper extraction ratio could be one ml 100% methanol for 100 µL of the liquid part of salmon feces or 100 mg of the solid part. The FCM directly detected in unextracted liquid part of feces correlated well with the FCM extracted from both liquid and solid part of the corresponding samples, without significant difference. Thus, it is feasible to measure FCM directly in the liquid part of salmon feces without any extraction procedure. Then, we applied this assay for FCM analysis in the group of salmon that experienced salmon pancreas disease (PD) and amoebic gill disease (AGD). We demonstrated 1) both plasma cortisol and FCM increased significantly during the outbreak of inflammatory disease (P < 0.01). Plasma cortisol level was elevated from 28 ± 40 ng/ml to 164.4 ± 62.5 ng/ml, FCM from 14.4 ± 13.2 ng/ml to 170.7 ± 89.7 ng/ml 2) Growth and starvation has no significant impact on either cortisol or FCM level. 3) FCM correlated well with plasma cortisol level (P < 0.01). Furthermore, there seems more individual variation in plasma cortisol levels than in FCM levels. These results suggest FCM could be directly analyzed in liquid part of salmon feces without extraction. This directly detected FCM level could represent the total fecal FCM level and plasma cortisol level. This simple and non-invasive method makes FCM a proper indicator for salmon welfare.

Exploring Diversity among Norwegian Borrelia Strains Originating from Ixodes ricinus Ticks.

Characterisation of Borrelia strains from Ixodes ricinus ticks is important in the epidemiological surveillance of vector-borne pathogens. Multilocus sequences analysis (MLSA) is a molecular genotyping tool with high discriminatory power that has been applied in evolutionary studies and for the characterisation of Borrelia genospecies. MLSA was used to study genetic variations in Borrelia strains isolated from I. ricinus ticks collected from the woodlands in Skodje. The results demonstrate that the 50 Borrelia strains were separated into 36 sequence types (STs) that were not previously represented in the MLST database. A distance matrix neighbour-joining tree (bootstrapped 500 iterations) showed four deeply branched clusters, and each deeply branched cluster represented one Borrelia genospecies. The mean pairwise genetic differences confirm the genospecies clustering. The combination of alleles separates the Borrelia strains from northwest Norway from the strains in the MLST database, thus identifying new STs. Although a highly divergent B. afzelii population could be expected, the heterogeneity among the B. garinii strains is more unusual. The present study indicates that the circulation of strains between migrating birds and stationary birds in this coastal region may play a role in the evolution of B. garinii strains.

Prevalence and Diversity among Anaplasma phagocytophilum Strains Originating from Ixodes ricinus Ticks from Northwest Norway.

The tick-borne pathogen Anaplasma phagocytophilum causes great concern for livestock farmers. Tick-borne fever is a widespread disease in Norway, and antibodies have been produced amongst sheep, roe deer, red deer, and moose. The main vector Ixodes ricinus is found along the Norwegian coastline as far north as the Arctic Circle. A total number of 1804 I. ricinus ticks were collected and the prevalence of the pathogen was determined by species-specific qPCR. The overall infection rate varied from 2.83% to 3.32%, but there were no significant differences (p = 0.01) in the overall infection rate in 2010, 2011, or 2012. A multilocus sequencing analysis was performed to further characterise the isolates. The genotyping of 27 strains resulted in classification into 19 different sequences types (ST), none of which was found in the MLST database. The nucleotide diversity was for every locus <0.01, and the number of SNPs was between 1 and 2.8 per 100 bp. The majority of SNPs were synonymous. A goeBURST analysis demonstrated that the strains from northwest Norway cluster together with other Norwegian strains in the MLST database and the strains that are included in this study constitute clonal complexes (CC) 9, 10, and 11 in addition to the singleton.

DGGE Identification of Microorganisms Associated with Borrelia burgdorferi Sensu Lato- or Anaplasma phagocytophilum-Infected Ixodes ricinus Ticks from Northwest Norway.

Ticks acquire a wide range of microorganisms as a natural part of their lifecycle. Bacteria, viruses, and protozoa can be transmitted to ticks during feeding and free-living phases. DGGE profiling is a molecular method to describe the microbial population associated with ticks and demonstrate some of the complexity and variety of tick-borne microorganisms. The present study profiled a total of 120 I. ricinus ticks, which were divided into three equally sized groups. We found that B. burgdorferi s.l.-infected ticks presented a pattern consisting of bacterial Pseudomonas spp. (67.5%), Bacillus spp. (50%), and Sphingomonas spp. (77.5%), while A. phagocytophilum-infected ticks were associated with Pseudomonas spp. (82.5%) and Sphingomonas spp. (57.5%). All profiles had one or more Pseudomonas species present, and the intramitochondrial endosymbiont Candidatus Midichloria mitochondrii was present in more than 25% of the samples. Statistical analysis demonstrated that the microbial communities were not significantly different between the groups and that the groups could not be characterised by a specific microbial population.

Prevalence of Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, and Borrelia valaisiana in Ixodes ricinus ticks from the northwest of Norway.

OBJECTIVES: Over a 3-y period, Ixodes ricinus ticks were randomly collected to study the prevalence of 4 Borrelia species: B. burgdorferi sensu stricto, B. afzelii, B. garinii and B. valaisiana. While B. burgdorferi s. s., B. afzelii, and B. garinii have been associated with human borreliosis in Norway for several years, B. valaisiana was reported in a Norwegian tick for the first time in 2010. METHODS: A real-time polymerase chain reaction (qPCR) was developed as an easy-to-use method, with high sensitivity and specificity, to detect and genospecies-type B. burgdorferi s. s., B. afzelii, B. garinii, and B. valaisiana in I. ricinus ticks. A combination of species-specific primers and TaqMan MGB probes labelled with fluorescents with different emission spectra, ensured a highly specific method with the potential to detect more than 1 genospecies in 1 run. Sequencing of the housekeeping gene recG from 48 Borrelia-positive samples was used to confirm specificity. Denaturing gradient gel electrophoresis profiling of tick-borne bacteria was used to help optimize the assay sensitivity. RESULTS: The qPCR assay was applied to analyze 1808 I. ricinus ticks collected in the field, which resulted in an overall infection rate of 14.8%, 18.7%, and 14.3% in 2010, 2011, and 2012, respectively. The majority of the Borrelia-infected ticks were infected with B. afzelii. CONCLUSIONS: The overall infection rate is somewhat lower than that reported in the most recent study of the infection rate in southern Norway in 2010, and this study indicates that the infection rate varies from one year to another.

Identification of bacteria infecting Ixodes ricinus ticks by 16S rDNA amplification and denaturing gradient gel electrophoresis.

Ticks harbor a complex microbial population, which they acquire while feeding on a variety of mammalians and birds. Zoonotic diseases transferred by ticks are an increasing problem and have become a burden to the community. 16S rDNA amplification and denaturing gradient gel electrophoresis (DGGE) enables detection of the broad spectrum of bacteria that settles in the ticks. Profiling the complete microbial population in ticks may provide a better understanding of the ticks' potential to harbor and disperse pathogens. Separation of pathogenic species by DGGE is based on variation in %GC content within the 16S rDNA genetic region. Sequencing of these fragments allows identification of bacterial species. Present study identified some well-known tick-infecting bacteria, such as members of genus Borrelia, Rickettsiales, and Pseudomonas, but also less described tick-infecting bacteria such as Rhodococcus erythropolis, Spiroplasma spp., and an endosymbiont of the microarthropod Folsomia candida. This is the first report of Segniliparus rugosus-infected Ixodes ricinus ticks. Also, it is the first report of several of these pathogens in the Norwegian tick population.