Koi herpesvirus (KHV) has infected farmed common carp in Poland clinically and asymptomatically since 2004. The role of non-carp species as vectors of virus transmission is well known except for in the case of KHV. The aim was to better understand this virus’ infection and transmission pathways in common carp, looking at the potential vector role of fishes kept with them. Eight species were experimentally infected with KHV by immersion in a suspension at 20°C ±1 and transferred to a tank after 45 minutes. Specimens were euthanised at intervals up to 56 days post infection (dpi) and tissue was examined for KHV DNA. Surviving infected fishes were introduced at intervals, each time into a separate tank, to naïve common carp for experimental infection. These were observed daily for symptoms, sacrificed along with controls after three months, and dissected to provide tissue samples. Also fish from 14 species collected from a farm with a history of KHV were sampled from 3 to 22 months after disease was confirmed. Organ sections from single fish were collected in a single tube. Viral DNA was detected in tench and roach samples up to 49 dpi, but in three-spined stickleback and stone maroko samples only up to 14 dpi. Transmission of KHV to naïve carp occurred after cohabitation. KHV DNA was detected in three fish species three months after the farm outbreak. We confirmed that grass and Prussian carp, tench, roach, and brown bullhead can transfer the virus to naïve common carp.

The Shewanella putrefaciens group are ubiquitous microorganisms recently isolated from different freshwater fish species and causing serious health disorders. The purpose of the study was to characterise isolates of the S. putrefaciens group with special emphasis on elucidating serological diversity and determining putative virulence factors. Isolates collected from freshwater fish (n = 44) and reference strains were used. The identification of bacteria was carried out using biochemical kits and 16S rRNA sequencing. Polyclonal antibodies were prepared against the S. putrefaciens group. The bacterium’s susceptibility to antimicrobial agents, its enzymatic properties, and its adhesion ability to fish cell lines were also tested. Finally, selected isolates were used in challenge experiments in common carp and rainbow trout. Excluding six isolates undeterminable for species, the bacteria were classified to three species: S. putrefaciens, S. xiamenensis, and S. oneidensis, and showed some phenotypic diversity. Fourteen serological variants of the S. putrefaciens group were determined with the newly developed serotyping scheme. Serodiversity may play an important role in the virulence of particular isolates. Further, S. putrefaciens group members adhere to epithelial cells and produce enzymes which may contribute to their virulence. Challenge tests confirmed the pathogenicity of the S. putrefaciens group for fish.

Introduction: Immune-potentiating functions of Lactobacillus plantarum strains in the common carp were evaluated. Material and Methods: Fourteen days of feeding fish dry diet supplemented with the bacteria provided parameters of nonspecific humoral immunity (lysozyme, ceruloplasmin, γ-globulin, total protein levels, and serum bactericidal activity) and cellular immunity (pinocytosis, respiratory burst activity, and potential killing activity of organ phagocytes), as well as the proliferative response of organ lymphocytes stimulated with mitogens. The resistance of fish to infection with Aeromonas hydrophila was also determined. Results: Dietary supplementation with L. plantarum had a substantial influence on the activity of organ phagocytes, especially the potential killing activity of head kidney cells. A significant increase in the proliferative activity of LPS-stimulated B lymphocytes and in the levels of γ-globulins and total protein was observed. The supplemented diet conveyed higher resistance than the control diet as the cumulative fish mortalities after infection with A. hydrophila were 65% and 85%, respectively. Conclusion: The results indicate that dietary supplementation with L. plantarum stimulates the antibacterial resistance of common carp and may reinforce defence against bacterial infections, but further studies need to be conducted.

Objective—To investigate safety and efficacy of a cyprinid herpesvirus type 3 (CyHV3) modified-live virus vaccine for the prevention of koi herpesvirus disease (KHVd). Animals—420 healthy koi (Cyprinus carpio koi). Procedures—Fish were vaccinated with a 1× dose or 10× overdose of CyHV3 modified-live virus vaccine or a placebo through bath exposure in tanks at 22°C. Horizontal transmission of vaccine virus was evaluated by commingling unvaccinated and vaccinated fish. Efficacy was evaluated by challenge exposure of vaccinated and naïve fish to a wild-type virus. Fish that died were submitted for quantitative PCR assay for CyHV3 and histologic evaluation. Results—The CyHV3 vaccine was safe and efficacious, even at a 10× overdose. Vaccine-associated mortality rate was inversely associated with body weight, with a cumulative mortality rate of 9.4% (18/192) in fish weighing ≤ 87 g and no deaths in fish weighing > 87 g (0/48). Horizontal transfer of vaccine virus from vaccinates to naïve fish was negligible. For efficacy, the vaccine provided a significant reduction in mortality rate after challenge exposure to a wild-type virus, with a prevented fraction of 0.83 versus the placebo control fish. Conclusions and Clinical Relevance—KHVd is highly contagious and commonly leads to deaths in 80% to 100% of exposed fish, representing a major threat to koi and common carp populations throughout the world. The CyHV3 modified-live virus vaccine had a favorable safety profile and was an effective vaccine for the control of KHVd in koi weighing > 87 g.

Objective—To evaluate the long-term protective immunity of a cyprinid herpesvirus 3 (CyHV3) vaccine in naïve koi (Cyprinus carpio koi). Animals—72 koi. Procedures—Vaccinated koi (n = 36) and unvaccinated control koi (36) were challenge exposed to a wild-type CyHV3 strain (KHVp8 F98-50) 13 months after vaccination. Results—The CyHV3 vaccine provided substantial protective immunity against challenge exposure. The proportional mortality rate was less in vaccinated koi (13/36 [36%]) than in unvaccinated koi (36/36 [100%]). For koi that died during the experiment, mean survival time was significantly greater in vaccinated than in unvaccinated fish (17 vs 10 days). Conclusions and Clinical Relevance—The CyHV3 vaccine provided substantial protective immunity against challenge exposure with CyHV3 13 months after vaccination. This provided evidence that koi can be vaccinated annually with the CyHV3 vaccine to significantly reduce mortality and morbidity rates associated with CyHV3 infection.

OBJECTIVE To describe a technique for endoscopic evaluation of the coelomic viscera of koi (Cyprinus carpio) and to evaluate the ability to visually examine coelomic structures by use of an approach cranial or caudal to the pelvic girdle. ANIMALS 16 subadult koi. PROCEDURES Koi were anesthetized with buffered tricaine methanesulfonate. Coelioscopic examination was performed via a ventral midline incisional approach cranial or caudal to the pelvic girdle. A 2.7-mm × 18-cm 30° oblique endoscope within a 4.8-mm operating sheath and infusion of saline (0.9% NaCl) solution was used. Ease of entry into the coelomic cavity and visual examination of structures were scored for each fish. Fish were euthanized 2 or 8 weeks after the procedure, and necropsy was performed. RESULTS The coelioscopic procedure was tolerated well, and all koi recovered uneventfully. For all fish, ease of entry and visual examination scores of the liver, intestines, gonads, heart, and anterior kidney were satisfactory to excellent. Visual examination of the posterior kidney and swim bladder was satisfactory to difficult, whereas the spleen and gallbladder were not visually identified. No significant differences were noted in entry or visual examination scores between the cranial and caudal approaches or between sexes. Minor complications included mild hemorrhage, rupture of the gonadal capsule, formation of adhesions between the viscera and incision site, and delayed healing of the incision. CONCLUSIONS AND CLINICAL RELEVANCE Diagnostic coelioscopy of koi appeared to be safe and effective. This procedure could have potential for use in examination of coelomic structures and disease diagnosis.

OBJECTIVE To determine in vivo effects of nitric oxide (NO) on blood-brain barrier (BBB) permeability in common carp (Cyprinus carpio L.). ANIMALS 148 carp. PROCEDURES Carp received glyceryl trinitrate (1 mg/kg) as an NO donor or received no treatment (control group). Nitrite and nitrate concentrations in carp sera were determined 0.25, 1, 3, 6, 8, 12, and 24 hours after treatment. In control and treatment groups, BBB permeability was analyzed by assessment of leakage of Evans blue dye into various brain areas at 6, 12, and 24 hours after glyceryl trinitrate treatment. Brain edema was determined by means of the wet-dry weight method and assessed with light microscopy on H&E-stained preparations of tissues obtained 6 and 24 hours after glyceryl trinitrate treatment. RESULTS Treatment with glyceryl trinitrate induced endogenous synthesis of NO, which was upregulated 6 and 8 hours after treatment. Increased NO synthesis was associated with increased permeability of the BBB, which developed 6 hours after treatment with the NO donor. Although the BBB became impermeable again by 12 hours after glycerol trinitrate treatment, brain edema still persisted 24 hours after treatment. CONCLUSIONS AND CLINICAL RELEVANCE In this study, treatment with an NO donor caused reversible opening of the BBB and brain edema in common carp. An intact BBB is important to prevent influx of potentially harmful substances into the brain. This investigation highlighted the possibility of BBB disarrangement caused by NO, a substance found in the CNS of all vertebrates evaluated.

OBJECTIVE To determine the pharmacokinetics of danofloxacin following IM administration of a single dose (10 mg/kg) in koi (Cyprinus carpio). ANIMALS 69 healthy adult koi housed in a 980-L flow-through-system tank. PROCEDURES 3 fish were kept as untreated controls, and the remaining 66 fish were assigned to 11 treatment groups with 6 fish/group. Fish in the treatment groups were given a single dose of danofloxacin (10 mg/kg) IM in the left epaxial musculature. Fifteen, 30, and 45 minutes and 1, 4, 12, 24, 72, 96, 120, and 144 hours after administration of danofloxacin, fish in each treatment group were euthanized, and blood samples and samples of liver, spleen, gill, anterior kidney, posterior kidney, skin and muscle, and scales were collected. Plasma and tissue drug concentrations were determined by liquid chromatography–tandem mass spectrometry, and noncompartmental pharmacokinetic analyses were performed. Tissues from the untreated control fish and fish euthanized 144 hours after danofloxacin administration were examined histologically. RESULTS Maximum plasma concentration (mean, 8,315.7 ng/mL) was reached approximately 45 minutes after danofloxacin administration; plasma elimination half-life was 15 hours. Danofloxacin was detected in all examined tissues from all 6 fish euthanized 15 minutes after drug administration and was detected in some tissues from 3 of the 6 fish euthanized 144 hours after drug administration. For all tissues, results of histologic examination were unremarkable. CONCLUSIONS AND CLINICAL RELEVANCE IM administration of a single dose (10 mg/kg) of danofloxacin in koi resulted in rapid absorption, with maximum plasma concentration reached approximately 45 minutes after drug administration; the drug could still be detected in some tissues 144 hours after administration.