Impact of Infection with Flavobacterium psychrophilum and Antimicrobial Treatment on the Intestinal Microbiota of Rainbow Trout.

The diversity and composition of intestinal microbiota in rainbow trout have been studied using next-generation sequencing (NGS), although few studies have examined the effects of antimicrobials. We evaluated the effect of antibiotics florfenicol and erythromycin and infection with or without Flavobacterium psychrophilum on the intestinal microbiota in rainbow trout juveniles (30-40 g) using NGS. Prophylactic oral antibiotic treatments were administered for 10 days before groups of fish were injected intraperitoneally with virulent F. psychrophilum. Intestinal content (allochthonous bacteria) was collected at day -11, 0, 12, and 24 p.i., and the v3-v4 region of the 16S rRNA gene was sequenced using Illumina MiSeq. Before prophylactic treatment, Tenericutes and Proteobacteria were the most abundant phyla identified and Mycoplasma was the most abundant genus. Fish infected with F. psychrophilum had decreased alpha diversity and a high abundance of Mycoplasma. Fish administered florfenicol had increased alpha diversity compared to the control at day 24 p.i., although both florfenicol and erythromycin-treated fish had a higher abundance of potential pathogens, specifically Aeromonas, Pseudomonas, and Acinetobacter. Mycoplasma disappeared after treatment but appeared again after day 24. This study demonstrates that prophylactic oral treatment with antibiotics florfenicol and erythromycin as well as F. psychrophilum infection changed the composition of intestinal microbiota in rainbow trout juveniles that did not recover by day 24 p.i. and further long-term effects on the host need to be investigated.

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens.

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.

Mass mortality associated with koi herpesvirus in wild common carp in Canada.

Koi herpesvirus (KHV) was identified as being associated with more than one mortality event affecting common carp in Canada. The first was an extensive mortality event that occurred in 2007 in the Kawartha Lakes region, Ontario, affecting Lakes Scugog and Pigeon. Fish had branchial necrosis and hepatic vasculitis with an equivocal interstitial nephritis. Several fish also had branchial columnaris. Subsequent mortality events occurred in 2008 in additional bodies of water in south-central Ontario, such as Lake Katchewanooka and outside of Ontario in Lake Manitoba, Manitoba. Koi herpesvirus was detected in fish submitted for examination from all of these lakes by polymerase chain reaction (PCR), and sequence of the PCR product revealed 100% homology to KHV strains U and I. Real-time PCR analysis of KHV-infected wild carp revealed viral loads ranging from 6.02×10(1) to 2.4×10(6) copies µg(-1) host DNA. This is the first report of KHV in Canada.

A prospective matched nested case-control study of bacterial gill disease outbreaks in Ontario, Canada government salmonid hatcheries.

Early-rearing salmonids in Ontario, Canada government fish hatcheries have been persistently affected by bacterial gill disease (BGD), and outbreaks at these locations have often been associated with high morbidity and mortality. The causative agent of BGD, Flavobacterium branchiophilum, is ubiquitous in fresh water, and outbreaks of BGD are considered to be associated with deleterious environmental conditions. This paper summarizes a 14-month rearing unit-level prospective nested matched case-control investigation at six Ontario government hatcheries (raising a total of six different salmonid species) to identify, and quantify the effects of, important predictors of BGD outbreaks. Ongoing husbandry data were collected on all early-rearing (<9 months of age) fish tank-lots ("tank-lot"=a group of fish from a specific lot existing in a single hatchery tank for a given period during the study time frame) at participating hatcheries, and all outbreaks of BGD were confirmed by light microscopy during the study period. Control tank-lots were selected at the end of the study and matched to individual cases based on time, hatchery, and species. Data were analyzed using logistic regression modeling, controlling for fish age. The final multivariable model indicated that affected tank-lots were significantly more likely to have had lower fish numbers, lower individual fish weights, higher mortality levels and higher feeding rates during the week preceding observed BGD outbreaks than were asymptomatic control tank-lots. Refinements in the observation and manipulation of these factors could therefore aid in the prevention of fish losses associated with observable BGD outbreaks. The predictive (as opposed to causal) nature of the identified factors needs to be considered, and further research is required to understand the relationships between these factors and BGD.

Rearing unit-level factors associated with bacterial gill disease treatment in two Ontario, Canada government salmonid hatcheries.

Early-rearing salmonids in Ontario Ministry of Natural Resources (OMNR) fish hatcheries have been consistently affected by bacterial gill disease (BGD) (causative agent: Flavobacterium branchiophilum) for many years. Separate retrospective epidemiological investigations of BGD treatments at two OMNR fish hatcheries (Hatcheries A and B) for the 1999 production year were conducted using on-site hatchery records. Both investigations were carried out at the rearing unit-level, with early-rearing (<9 months of age) "tank-lot" as the unit of analysis to identify unique fish populations over time. Multivariable repeated measures logistic regression models were created for both hatchery datasets, controlling for lot-level and species effects. For Hatchery A, the species brook trout (Salvelinus fontinalis) and brown trout (Salmo trutta) were significantly associated with BGD treatment, as well as lower water exchange rate, and higher feeding and mortality percentages during the 2 weeks previous to BGD treatment. At Hatchery B, the species brook trout (S. fontinalis) and splake (Salvelinus namaycush x S. fontinalis) were significantly associated with BGD treatment, as well as lower individual fish weights and treatment for BGD during the previous week. These results emphasize the importance of water quality, feeding rate, fish size and prior mortality on the development of BGD. Significant hatchery and species effects were evident, and future observational research on BGD must account for these factors in their design and analysis.

Factors associated with the incidence of bacterial gill disease in salmonid lots reared in Ontario, Canada government hatcheries.

Bacterial gill disease (BGD) (causative agent: Flavobacterium branchiophilum) has been a persistent problem in early-rearing salmonids in the Ontario Ministry of Natural Resources (OMNR) fish hatchery system. Retrospective epidemiological investigations of BGD diagnoses and treatments in OMNR fish hatcheries during the period 1991-2001 were conducted using University of Guelph Fish Health Laboratory and OMNR central office data. All investigations were conducted at the lot-level, which is the major within-hatchery-level of population aggregation. Survivorship of BGD diagnosis in early-rearing lots within seven individual hatcheries ranged from 84.2 to 100%; within individual species groups, survivorship was lowest (84.6%) in brook trout (Salvelinus fontinalis) lots. Annual risk percentages (cumulative incidence) for BGD diagnosis within hatchery and species groups varied considerably among years. Multivariable proportional hazards survival analysis indicated that the species brook trout, and the Spring (March-May) season, were significantly associated with treatment for BGD. Combined, these results emphasize the importance of hatchery, species, and time on the development of BGD. Future observational research on this disease must consider these factors in their design and analysis.