Evaluation of oxytetracycline leaching from pregelatinized starch-coated medicated fish feed.

The rapid growth and intensification of aquaculture industries have led to an increased use of antibiotics. Consequently, growing concerns have mounted over the environmental contamination of these drugs from medicated feeds and the risk that this poses for antimicrobial resistance. To circumvent environmental leaching, farmers topcoat medicated feeds with oil; however, this only partially addresses the issue. This study investigated the potential of food-grade pregelatinized corn starch (PGS) as a second top-coating agent to reduce oxytetracycline (OTC) leaching from the hand-mixed medicated feed. We immersed top-coated medicated feeds for different periods of time and measured the concentration of OTC in the water to determine leaching. We found a significantly lower level of OTC in water samples collected from the PGS-coated medicated feed compared to the non-PGS-coated medicated feed, with concentrations of OTC approximately 4 and 2.6 times the latter after 5 min and 2 h of water immersion, respectively. We also fed PGS-coated antibiotic feed to jade perch to determine if fish accepted the top-coating and whether they absorbed the OTC. Results from a feeding trial suggested no difference in palatability between PGS and non-PGS-coated medicated feed. We also found that muscle tissue from fish fed with the aforementioned diets had similar levels of OTC concentrations, suggesting that PGS coating does not alter the gastrointestinal absorption of this medication. From our experiment, we conclude that PGS is potentially a new top-coating agent to reduce leaching in hand-mixed OTC medicated feed.

Differential characterization of emerging skin diseases of rainbow trout--a standardized approach to capturing disease characteristics and development of case definitions.

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example.

Parasites and hepatic lesions among pink salmon, Oncorhynchus gorbuscha (Walbaum), during early seawater residence.

Juvenile pink salmon, Oncorhynchus gorbuscha (Walbaum), in the Broughton Archipelago region of western Canada were surveyed over 2 years for sea lice (Lepeophtheirus salmonis and Caligus clemensi), gross and microscopic lesions and evidence of infections with viruses and bacteria. The 1071 fish examined had an approximate ocean residence time no longer than 3 months. A high prevalence of degenerative liver lesions, renal myxosporean parasites and a low prevalence of skin lesions and sea lice were observed. No indications of viral or bacterial diseases were detected in either year. The monthly prevalence of sea lice in 2007 (18-51%) was higher than in 2008 (1-26%), and the infestation density exceeded the lethal threshold in only two fish. Degenerative hepatic lesions and renal myxosporean parasites occurred in approximately 40% of the pink salmon examined in June of both years, and the peak monthly prevalence of hepatocellular hydropic degeneration was greater in 2007 (32%, in May) than in 2008 (12%, in June). Logistic regression analysis found skin lesions and hepatocellular hydropic degeneration significantly associated with sea lice. Most parasites and lesions occurred during both years, but the prevalence was often higher in 2007. Fish weight was 35% less in June 2007 than in June 2008, but condition factor was not different. Further research is required to monitor inter-annual variations and aetiology of the liver lesions and to assess their potential role on pink salmon survival.

Oral DNA vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), against infectious haematopoietic necrosis virus using PLGA [Poly(D,L-Lactic-Co-Glycolic Acid)] nanoparticles.

A DNA vaccine against infectious haematopoietic necrosis virus (IHNV) is effective at protecting rainbow trout, Oncorhynchus mykiss, against disease, but intramuscular injection is required and makes the vaccine impractical for use in the freshwater rainbow trout farming industry. Poly (D,L-lactic-co-glycolic acid) (PLGA) is a U.S. Food and Drug Administration (FDA) approved polymer that can be used to deliver DNA vaccines. We evaluated the in vivo absorption of PLGA nanoparticles containing coumarin-6 when added to a fish food pellet. We demonstrated that rainbow trout will eat PLGA nanoparticle coated feed and that these nanoparticles can be detected in the epithelial cells of the lower intestine within 96 h after feeding. We also detected low levels of gene expression and anti-IHNV neutralizing antibodies when fish were fed or intubated with PLGA nanoparticles containing IHNV G gene plasmid. A virus challenge evaluation suggested a slight increase in survival at 6 weeks post-vaccination in fish that received a high dose of the oral vaccine, but there was no difference when additional fish were challenged at 10 weeks post-vaccination. The results of this study suggest that it is possible to induce an immune response using an orally delivered DNA vaccine, but the current system needs improvement.

Antibody response of two populations of common carp, Cyprinus carpio L., exposed to koi herpesvirus.

Common carp, Cyprinus carpio L., exposed to koi herpesvirus (KHV) may become persistently infected and populations containing such virus-infected individuals may transmit the virus to other fish when co-habited. Detection of virus-infected fish in a population is thus critical to surveillance and control programmes for KHV. A study was therefore designed to detect anti-KHV serum antibodies, with an enzyme-linked immunosorbent assay, in common carp following experimental exposures to KHV under varying environmental conditions. The study determined that a proportion of fish within a population experimentally exposed to KHV (at least 10-25%) develop high antibody titres (1/1600 or greater) to the virus, and this immunological response was detectable for several months (observed at the termination of the experiments at 65, 46 and 27 weeks post-exposure). Furthermore, this response was detected in one population of fish that did not succumb to a high level of mortality when maintained at water temperatures that were non-permissive for KHV. Elevating the water temperatures to permissive conditions for KHV resulted in recurrence of disease despite the presence of anti-virus antibodies, suggesting that serum antibodies alone are not protective under the conditions of our trials.

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio.

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.

An assessment of the variation in the prevalence of renal myxosporidiosis and hepatitis in wild brown trout, Salmo trutta L., within and between rivers in South-West England.

The prevalence of renal myxosporidiosis in wild brown trout, Salmo trutta, in seven river catchments in South-West England was investigated. Three hundred and twenty-seven fish were sampled from 16 sites, of which 54 (16.5%) were found, by histological examination of the kidney, to be infected with Tetracapsuloides bryosalmonae, the causative agent of proliferative kidney disease. No T. bryosalmonae infected fish were found in one river catchment, in other catchments the prevalence ranged from 2.5% to 36%. Hepatitis was strongly associated with the presence of T. bryosalmonae (odds ratio = 20.2, P < 0.001). Chloromyxum schurovi was found in 25% of fish and in six of seven river catchments, where the prevalence ranged from 2.4% to 63%. There was a strong negative association between the presence of T. bryosalmonae and C. schurovi (odds ratio = 0.10, P < 0.001). A hierarchical binomal model of the variance indicated that for T. bryosalmonae most of the variance existed at the site level, whereas for C. schurovi most variance existed at the river catchment level, suggesting that prevalence of T. bryosalmonae infection is determined largely by site level factors (e.g. presence of alternate host). The intraclass correlation coefficients (ICC) were 0.2 and 0.4 for T. bryosalmonae and C. schurovi, respectively, indicating the latter has higher effective transmission because of a higher level of infectiousness and/or abundance of alternate oligochaete hosts. These values can be used in future studies to estimate the sample sizes required to generate prevalence estimates with the required precision.

Assessing concentration of antibiotics in tissue during oral treatments against piscirickettsiosis.

The use of antimicrobials in aquaculture is increasingly being scrutinized. In Chile, piscirickettsiosis accounts for approximately 90% of the total volume of antibiotics used in marine aquaculture. Treatment failures are frequently reported, but there is limited information on why this occurs. Fish producers have started assessing the level of antibiotics in fish tissues during and immediately after in-feed treatments to determine if they are adequately medicating their fish. In this study, we evaluated the probability of finding antibiotic concentrations in muscle tissue above the minimum inhibitory concentration for 90% of the P. salmonis isolates (MIC90) recently tested in Chile, for two antibiotics commonly used in aquaculture. We found that the proportion of fish with antibiotic concentrations above the MIC90 varied, depending on the product used, species, day of sample collection, and size category of fish within a cage. The proportion of fish above the MIC90 was lower in fish treated with florfenicol than in fish treated with oxytetracycline. Using a mixed-effects logistic model, we modeled the probability of antibiotic concentrations above MIC90 when fish were treated with florfenicol. Our model suggested lower probabilities of having concentrations above MIC90 in Atlantic salmon than in rainbow trout when samples were collected 14 days after the treatment started compared to 7 days, and in the smaller fish within a cage. We discuss these findings and hypothesize about potential issues with treating large populations of fish with in-feed antimicrobials.

Tool for predicting Caligus rogercresseyi abundance on salt water salmon farms in Chile.

Caligus rogercresseyi is a host-dependent parasite that affects rainbow trout and Atlantic salmon in Chile. Numbers of sea lice on fish increase over time at relatively predictable rates when the environment is conducive to the parasite's survival and fish are not undergoing treatment. We developed a tool for the salmon industry in Chile that predicts the abundance of adult sea lice over time on farms that are relatively isolated. We used data on sea louse abundance collected through the SalmonChile INTESAL sea lice monitoring program to create series of weekly lice counts between lice treatment events on isolated farms. We defined isolated farms as those with no known neighbors within a 10 km seaway distance and no more than two neighbors within a 20 km seaway distance. We defined the time between sea lice treatments as starting the week immediately post treatment and ending the week before a subsequent treatment. Our final dataset of isolated farms consisted of 65 series from 32 farms, between 2009 and 2015. Given an observed abundance at time t = 0, we built a model that predicted 8 consecutive weekly sea louse abundance levels, based on the preceding week's lice prediction. We calibrated the parameters in our model on a randomly selected subset of training data, choosing the parameter combinations that minimized the absolute difference between the predicted and observed sea louse abundance values. We validated the parameters on the remaining, unseen, subset of data. We encoded our model and made it available as a Web-accessible applet for producers. We determined a threshold, based on the upper 97.5% predictive interval, as a guideline for producers using the tool. We hypothesize that if farms exceed this threshold, especially if the sea lice levels are above this threshold 2 and 4 weeks into the model predictions, the sea louse population on the farm is likely influenced by sources other than lice within the farm.

Evaluating the effect of synchronized sea lice treatments in Chile.

The sea louse is considered an important ectoparasite that affects farmed salmonids around the world. Sea lice control relies heavily on pharmacological treatments in several salmon-producing countries, including Chile. Among options for drug administration, immersion treatments represent the majority of antiparasitic control strategies used in Chile. As a topical procedure, immersion treatments do not induce a long lasting effect; therefore, re-infestation from neighbouring farms may undermine their efficacy. Synchronization of treatments has been proposed as a strategy to improve immersion treatment performance, but it has not been evaluated so far. Using a repeated-measures linear mixed-effect model, we evaluated the impact of treatment synchronization of neighbouring farms (within 10km seaway distance) on the adult lice mean abundance from weeks 2 to 8 post-treatment on rainbow trout and Atlantic salmon farms in Chile, while controlling for external and internal sources of lice before the treatments, and also for environmental and fish-related variables. Results indicate that treatment synchronization was significantly associated with lower adult lice levels from weeks 5 to 7 after treatment. This relationship appeared to be linear, suggesting that higher levels of synchronization may result in lower adult sea lice levels during these weeks. These findings suggest that synchronization can improve the performance of immersion delousing treatments by keeping sea lice levels low for a longer period of time. Our results may be applicable to other regions of the world where immersion treatments are widely used.

Fin erosion on rainbow trout on commercial trout farms in the United Kingdom.

Fish weighing less than 30 g and more than 100 g were sampled from 38 rearing units on 10 commercial farms growing rainbow trout for the table market. A fin index was calculated for each of the eight rayed fins on 949 trout by dividing their length by the standard length of the fish. There was a large range in the indices of all eight fins. The fin indices of the small and large fish were compared, controlling for farm effect. With the exception of the dorsal fin, all the indices were larger for the small fish than for the large fish, but the magnitude of the difference was greater for some fins than others. In comparison with the fins of wild fish, the pectoral and dorsal fins appeared to be most eroded and the damage to these fins was evident even in the small fish. The erosion of the caudal, anal and ventral (or pelvic) fins was more prominent in the larger fish. Variations in the fin indices of the caudal, anal and ventral fins suggested that there was little variation between rearing units on the same farm, but that there was significant variation between individual fish in the same rearing units, and between fish on different farms.

Reactivation of koi herpesvirus infections in common carp Cyprinus carpio.

Two co-habitation studies with common carp were conducted to determine whether latent infections of koi herpesvirus (KHV) exist. Fish were exposed to KHV using 2 different temperature profiles, which induced low and high initial mortality. Subsequently, certain groups of fish were co-habited with naive fish while others were not. Koi herpesvirus was reactivated in fish from 3 of the 5 experimental tanks. Reactivation of the virus occurred regardless of the initial mortality associated with the virus or whether fish were co-habited with naive fish. The reactivation of the virus in our experiments occurred several months after the initial exposure to KHV and appeared to be temperature dependent.

The effects of indalpine--a selective inhibitor of 5-HT uptake--on rat paradoxical sleep.

In order to document the role of monoamines in the reduction of paradoxical sleep by antidepressant drugs, we examined the effect of indalpine , a selective inhibitor of serotonin uptake. Indalpine dose dependently decreased paradoxical sleep and delayed its first appearance. Pretreatment with parachlorophenylalanine markedly decreased the effect of indalpine . In contrast, pretreatment with alpha-methylparatyrosine potentiated the indalpine -induced depression of paradoxical sleep. The results of the study indicate that the increase of extracellular concentration of 5-HT has an inhibitory effect on paradoxical sleep, and this effect is enlarged if catecholaminergic activity is reduced.

Substance P neuronal cell bodies in the human brain: complete mapping by immunohistofluorescence.

The localization of substance P-immunoreactive (SP-IR) cells was studied in 5 postmortem brains from aged human subjects using technical improvements combined with the indirect immunofluorescence method. The presence of various forms of SP-IR cells was observed in paleocortical and neocortical areas as well as in the basal ganglia, the brainstem and the spinal cord. The distribution of these SP-IR cell bodies was transposed onto our previous mapping of SP-IR fibers and terminals. The morphological differences between SP-containing cell bodies or areas of human and rat central nervous systems and the SP-changes in various states of human pathology are shortly discussed.

Simultaneous determinations of tolbutamide and its hydroxy and carboxy metabolites in serum and urine: application to pharmacokinetic studies of tolbutamide in the rat.

Methods of analysis of tolbutamide (1) and its hydroxylated (2) and carboxylated (3) metabolites in serum and urine based on high-performance liquid chromatography were developed. The separation was performed on a Apex ODS column in the isocratic mode using a mobile phase composed of 22.5% acetonitrile, 77.5% Sorensen phosphate buffer (pH 7.0), and 0.30 mL of tetrabutylammonium phosphate reagent (Pic A). The compounds were detected at 254 mm. The retention times of 3, 2, 1, and the internal standard chlorpropamide were 3.1, 4.1, 14.8, and 10.0 min, respectively. These conditions were suitable for the simultaneous quantitation of 1, 2, and 3 in serum or plasma samples, but not for the determination of metabolites 2 and 3 in urine. For the analysis of 2 and 3 in urine, the mobile phase was modified to 18% acetonitrile, 82% Sorensen phosphate buffer (pH 7.0), and 0.35 mL of Pic A. Under these conditions, the retention times of the carboxy and hydroxylated metabolites and the internal standard salicylic acid were 4.6, 6.7, and 8.1 min, respectively. These methods were applied to study the pharmacokinetics of 1 administered intravenously and intraperitoneally to the rat. Tolbutamide was almost completely recovered as metabolites 2 and 3 in the urine within 24 h.

Infectious hematopoietic necrosis virus antibody profiles in naturally and experimentally infected Atlantic salmon Salmo salar.

Atlantic salmon Salmo salar naturally and experimentally exposed to infectious hematopoietic necrosis virus (IHNV) in British Columbia, Canada, developed antibodies against the virus. More than 50% of the fish exposed to IHNV remained seropositive for several months after the IHN epizootic had subsided. The virus itself could not be detected in asymptomatic fish once the fish had recovered from IHN. The persistence of IHNV-specific antibodies in a large percentage of Atlantic salmon, from 4 different populations that survived an outbreak of IHN, and the lack of IHNV-specific antibodies in fish with no history of the disease, suggests that serology may be a useful tool for determining previous exposure to the virus. It may be important to determine whether Atlantic salmon have been infected with IHNV because, although the virus is difficult to detect in asymptomatic fish, an incidental finding suggests it may persist in a small number of fish after the outbreak has subsided. Furthermore, the presence of seropositive fish would be an indication that the virus may be enzootic at a farm, and such information would thus aid producers with stocking decisions.

Evidence for a carrier state of infectious hematopoietic necrosis virus in chinook salmon Oncorhynchus tshawytscha.

In British Columbia, Canada, infectious hematopoietic necrosis virus (IHNV) is prevalent in wild sockeye salmon Oncorhynchus nerka and has caused disease in seawater net-pen reared Atlantic salmon Salmo salar. In this study, chinook salmon Oncorhynchus tshawytscha experimentally exposed to an isolate of IHNV found in British Columbia became carriers of the virus. When Atlantic salmon were cohabited with these virus-exposed chinook salmon, IHNV was isolated from the Atlantic salmon. Identification of chinook salmon populations that have been exposed to IHNV may be difficult, as virus isolation was successful only in fish that were concurrently infected with either Renibacterium salmoninarum or Piscirickettisia salmonis. Also, IHNV-specific antibodies were detected in only 2 of the 70 fish experimentally exposed to the virus. Two samples collected from chinook salmon exposed to IHNV while at a salt water net-pen site had a seroprevalence of 19 and 22%; however, the inconsistencies between our laboratory and field data suggest that further research is required before we can rely on serological analysis for identifying potential carrier populations. Because of the difficulty in determining the exposure status of populations of chinook salmon, especially if there is no concurrent disease, it may be prudent not to cohabit Atlantic salmon with chinook salmon on a farm if there is any possibility that the latter have been exposed to the virus.

Understanding sources of sea lice for salmon farms in Chile.

The decline of fisheries over recent decades and a growing human population has coincided with an increase in aquaculture production. As farmed fish densities increase, so have their rates of infectious diseases, as predicted by the theory of density-dependent disease transmission. One of the pathogen that has increased with the growth of salmon farming is sea lice. Effective management of this pathogen requires an understanding of the spatial scale of transmission. We used a two-part multi-scale model to account for the zero-inflated data observed in weekly sea lice abundance levels on rainbow trout and Atlantic salmon farms in Chile, and to assess internal (farm) and external (regional) sources of sea lice infection. We observed that the level of juvenile sea lice was higher on farms that were closer to processing plants with fish holding facilities. Further, evidence for sea lice exposure from the surrounding area was supported by a strong positive correlation between the level of juvenile sea lice on a farm and the number of gravid females on neighboring farms within 30 km two weeks prior. The relationship between external sources of sea lice from neighboring farms and juvenile sea lice on a farm was one of the strongest detected in our multivariable model. Our findings suggest that the management of sea lice should be coordinated between farms and should include all farms and processing plants with holding facilities within a relatively large geographic area. Understanding the contribution of pathogens on a farm from different sources is an important step in developing effective control strategies.

Antimicrobial use and resistance in aquaculture: findings of a globally administered survey of aquaculture-allied professionals.

There is limited published information regarding antimicrobial use (AMU) and antimicrobial resistance (AMR) in aquaculture. Our objective was to determine the opinions of aquaculture-allied professionals around the world on the frequency of AMU and AMR in common aquatic species. The study questionnaire included five sections: respondent demographics, extent of AMU in aquaculture, frequency of observations of AMR in aquaculture, AMR monitoring and surveillance and antimicrobial susceptibility testing in various jurisdictions. It was administered in English and Spanish to 604 professionals in 25 countries and with varying expertise in aquaculture. The response rate was 33% (199/604). Over half of the participants had >10 years of experience in aquaculture: 70% (140/199) were involved in fish health/clinical work and their primary experience was with salmon, tilapia, trout, shrimp (including prawn) and/or catfish. Tetracycline use was reported by 28%, 46%, 18%, 37% and 9% of respondents working with catfish, salmon, tilapia, trout and shrimp, respectively. Resistance to tetracycline in one or more species of bacteria was reported as 'frequent-to-almost always' for the same aquaculture species by 39%, 28%, 17%, 52% and 36% of respondents, respectively. 'Frequent-to-almost always' use of quinolone was reported by 70% (32/46) and 67% (8/12) of respondents from the United States and Canada, respectively, where quinolone products are not approved for aquaculture, and extra-label fluoroquinolone use is either prohibited (United States) or discouraged (Canada). Similar frequencies of quinolone use were also reported by the majority of respondents from Europe [70% (7/10)] and Asia [90% (9/10)] where labelled indications exist. This baseline information can be used to prioritize research or surveillance for AMU and AMR in aquaculture.