Effects of Salinity on Yersinia ruckeri Infection of Rainbow Trout and Brown Trout

Abstract

Juvenile rainbow trout Oncorhynchus mykiss and brown trout Salmo trutta acclimated to freshwater or salinities of 9.0‰ or less were exposed to Yersinia ruckeri, the bacterial pathogen that causes enteric redmouth disease (ERM). Both species of fish were kept in the same recirculating systems after bacterial exposure. Rainbow trout mortality was significantly (P < 0.05) different in each salinity: 96.5% in freshwater, 89.5% in water of 1.1‰ salinity, 81.3% in 3.0‰ salinity, and 75.0% in 9.0‰ salinity (model SE = 1.0). All deaths occurred between 3 and 12 d after exposure to Y. ruckeri. Only 2.3% of brown trout in all salinities died, and differences among treatments were not significant. For both fish species, Y. ruckeri was isolated from liver, spleen, and trunk kidney of fish dying during this experiment, and lesions of rainbow trout were consistent with ERM. Yersinia ruckeri was not isolated from brown trout surviving for 21 d after bacterial exposure but was isolated from 3 of 24 surviving rainbow trout; a polymerase chain reaction assay detected the DNA of Y. ruckeri in 3 additional rainbow trout survivors. Neither the lesions of fish with ERM nor the percentage of surviving fish subclinically infected with Y. ruckeri was affected by salinity. Bacterial growth in vitro was not affected by low (≤9.0‰) salinity; however, bacterial adhesion to polystyrene was significantly reduced as salinity increased. Although mortality caused by Y. ruckeri was significantly lower for rainbow trout in water with slightly increased salinity, none of the salinities tested was effective in preventing serious losses caused by this pathogen in recirculating systems.     

Efficacy of lipopolysaccharide antigen of Yersinia ruckeri in rainbow trout by intraperitoneal and bath immersion administration

In this study, Intraperitoneal (IP) and bath immersion (BI) vaccine trials were conducted in fish with a mean weight of 6.3 g. Rainbow trout vaccinated with lipopolysaccharide (LPS) was 50 mg/L protein concentration and challenged by IP injection with 9.8 × 10⁶ cell/ml of Yersinia ruckeri at 45 days post-immunization had a relative percent survival (RPS). To obtain an effective bath immersion vaccine against yersiniosis, LPS preparation was obtained from the Y. ruckeri and with the LPS antigen. After 28 and 60 days vaccinated fish with first and second immunizations by LPS were challenged via intraperitoneal injection with 9.8 × 10⁶ cell/ml of Y. ruckeri for evaluating the mortality rates and calculating the relative percentage of survival (RPS). RPS value of experimental groups, which was significantly (P < 0.05) larger than that of the control group.     

Comparative protection of two different commercial vaccines against Yersinia ruckeri serotype O1 and biotype 2 in rainbow trout (Oncorhynchus mykiss)

Differentially extended specific protection by two commercial vaccines against Yersinia ruckeri serotype O1 biotype 2 was studied following 30 s immersion exposure. Rainbow trout were challenged intra-peritoneally (i.p.) with Y. ruckeri serotype O1, biotype 2 (≈10⁶ to 10⁷ CFU/fish) at 4, 6 and 8 months after vaccination with vaccines containing either biotype 1 (AquaVac^® ERM) or both biotypes 1 and 2 (AquaVac^® RELERA?). The specific pattern of vaccine-mediated protection was evaluated by relative percentage survival (RPS) analysis at 4 and 6 months post-vaccination and by obtaining gross pathological observations at 4 and 8 months respectively. We determined specific significant and superior protection in terms of increased survivability in AquaVac^® RELERA? vaccinated fish and observed correspondingly fewer pathological changes. The challenge trials indicated a longer protection for at least 6 months without any booster vaccination. A specific and adaptive response induced by AquaVac^® RELERA? vaccine against Y. ruckeri biotype 2 was clearly indicated. In addition, some degree of cross protection rendered by AquaVac^® ERM containing biotype 1 during infection with Y. ruckeri biotype 2 was also noted.     

Survivability of Infectious Hematopoietic Necrosis Virus in Fertilized Eggs of Masu and Chum Salmon

Abstract

The possibility of vertical transmission of infectious hematopoietic necrosis virus (IHNV) was studied with the eggs of masu (cherry) salmon Oncorhynchus masou and chum salmon O. keta. The surfaces of eggs and sperm were contaminated with IHNV (10^3.8-10^4.8 50% tissue culture infective dose [TCID50]/egg) and then the eggs were fertilized. Eggs just after fertilization and embryonated eggs also were infected by injection with IHNV (10^3.8 TCID50/egg) directly into the yolk. During incubation, eggs were held in running water at 10°C. Mortality of the eggs or hatched progeny was determined and isolation of IHNV on the surface or inside of the eggs was determined during the incubation period. No mortality occurred and no virus was detected in fertile eggs from contaminated gametes. For injected eggs, IHNV was not detected on the surface of masu and chum salmon eggs after 1 d of incubation. Infectivity of IHNV inside the eggs decreased gradually and could not be detected after 1 month of incubation. This rate of IHNV reduction in the fertilized egg was similar to that found in a mixture of IHNV and homogenized yolk contents. Several individual yolk components also showed anti-IHNV activity. When eyed eggs were injected with IHNV, the embryos of both masu and chum salmon became infected, and the concentration of virus increased rapidly and reached more than 10^6.5 TCID50/fish. The cumulative mortality of eggs injected at the eyed stage for both masu and chum salmon was 90%. The susceptibilities of hatched-out larvae of masu and chum salmon to IHNV were different; cumulative mortality was more than 90% in masu salmon and 20–30% in chum salmon artificially infected with the virus. We concluded that vertical transmission of IHNV is doubtful because the virus is apparently unable to survive in eggs before the eyed stage.     

Hemodynamic,respiratory and sedative effects of progressively increasing doses of acepromazine in conscious dogs

ObjectiveTo evaluate the effects of progressively increasing doses of acepromazine on cardiopulmonary variables and sedation in conscious dogs.Study designProspective, experimental study.AnimalsA group of six healthy, adult, mixed-breed dogs weighing 16.5 ± 5.0 kg (mean ± standard deviation).MethodsDogs were instrumented with thermodilution and arterial catheters for evaluation of hemodynamics and arterial blood gases. On a single occasion, acepromazine was administered intravenously to each dog at 10, 15, 25 and 50 μg kg^–1 at 20 minute intervals, resulting in cumulative acepromazine doses of 10 μg kg^–1 (ACP₁₀), 25 μg kg^–1 (ACP₂₅), 50 μg kg^–1 (ACP₅₀) and 100 μg kg^–1 (ACP₁₀₀). Hemodynamic data and sedation scores were recorded before (baseline) and 20 minutes after each acepromazine dose.ResultsCompared with baseline, all acepromazine doses significantly decreased stroke index (SI), mean arterial pressure (MAP) and arterial oxygen content (CaO₂) with maximum decreases of 16%, 17% and 21%, respectively. Cardiac index (CI) decreased by up to 19% but not significantly. Decreases of 26–38% were recorded for oxygen delivery index (DO₂I), with significant differences for ACP₅₀ and ACP₁₀₀. Systemic vascular resistance index (SVRI) and heart rate did not change significantly. No significant difference was found among acepromazine doses for hemodynamic data. After ACP₁₀, mild sedation was observed in five/six dogs and moderate sedation in one/six dogs, whereas after ACP₂₅, ACP₅₀ and ACP₁₀₀, moderate sedation was observed in five/six or six/six dogs.Conclusions and clinical relevanceIn conscious dogs, acepromazine decreased MAP, SI, CaO₂ and DO₂I, but no significant dose effect was detected. SVRI was not significantly changed, suggesting that the reduction in MAP resulted from decreased CI. The ACP₂₅, ACP₅₀ and ACP₁₀₀ doses resulted in moderate sedation in most dogs; ACP₁₀ resulted in only mild sedation.     

Yersinia ruckeri,the causative agent of enteric redmouth disease in fish

Enteric redmouth disease (ERM) is a serious septicemic bacterial disease of salmonid fish species. It is caused by Yersinia ruckeri, a Gram-negative rod-shaped enterobacterium. It has a wide host range, broad geographical distribution, and causes significant economic losses in the fish aquaculture industry. The disease gets its name from the subcutaneous hemorrhages, it can cause at the corners of the mouth and in gums and tongue. Other clinical signs include exophthalmia, darkening of the skin, splenomegaly and inflammation of the lower intestine with accumulation of thick yellow fluid. The bacterium enters the fish via the secondary gill lamellae and from there it spreads to the blood and internal organs. Y. ruckeri can be detected by conventional biochemical, serological and molecular methods. Its genome is 3.7 Mb with 3406–3530 coding sequences. Several important virulence factors of Y. ruckeri have been discovered, including haemolyin YhlA and metalloprotease Yrp1. Both non-specific and specific immune responses of fish during the course of Y. ruckeri infection have been well characterized. Several methods of vaccination have been developed for controlling both biotype 1 and biotype 2 Y. ruckeri strains in fish. This review summarizes the current state of knowledge regarding enteric redmouth disease and Y. ruckeri: diagnosis, genome, virulence factors, interaction with the host immune responses, and the development of vaccines against this pathogen.     

Hemodynamic effects of incremental doses of acepromazine in isoflurane-anesthetized dogs

ObjectiveTo evaluate the effects of incremental doses of acepromazine on hemodynamics in isoflurane-anesthetized dogs.Study designProspective, experimental study.AnimalsHealthy, adult, mixed-breed dogs (two male and four female) weighing 16.8 ± 5.1 kg (mean ± standard deviation).MethodsDogs were anesthetized with propofol (7 mg kg^–1) intravenously (IV) and isoflurane. Thermodilution and arterial catheters were placed for hemodynamic monitoring and arterial blood sampling for blood gas analysis. Baseline measurements were performed with stable expired concentration of isoflurane (Fe′Iso) at 1.8%. Each dog was then administered four incremental acepromazine injections (10, 15, 25 and 50 μg kg^–1) IV, and measurements were repeated 20 minutes after each acepromazine injection with Fe′Iso decreased to 1.2%. The four acepromazine injections resulted in cumulative doses of 10, 25, 50 and 100 μg kg^–1 (time points ACP₁₀, ACP₂₅, ACP₅₀ and ACP₁₀₀, respectively).ResultsCompared with baseline, cardiac index (CI) increased significantly by 34%, whereas systemic vascular resistance index (SVRI) decreased by 25% at ACP₅₀ and ACP₁₀₀. Arterial oxygen content (CaO₂) was significantly lower than baseline after all acepromazine injections (maximum decreases of 11%) and was lower at ACP₅₀ and ACP₁₀₀ than at ACP₁₀. No significant change was found in heart rate, stroke index, oxygen delivery index and systolic, mean and diastolic blood pressures. Hypotension (mean arterial pressure < 60 mmHg) was observed in one dog at baseline, ACP₁₀, ACP₂₅ and ACP₁₀₀, and in two dogs at ACP₅₀.Conclusions and clinical relevanceCompared with isoflurane alone, anesthesia with acepromazine–isoflurane resulted in increased CI and decreased SVRI and CaO₂ values. These effects were dose-related, being more pronounced at ACP₅₀ and ACP₁₀₀. Under the conditions of this study, acepromazine administration did not change blood pressure.     

Experimental Infection of Brook Trout with Infectious Hematopoietic Necrosis Virus Types 1 and 2

Abstract

Fry of brook trout Salvelinus fontinalis became infected and diseased after immersion exposure to infectious hematopoietic necrosis virus (IHNV), but a long-lasting IHNV carrier state was not induced. Duplicate groups of 100 fish were immersed for 6 h in baths containing a type 1 (Round Butte, RB) or a type 2 (Rangen, RA) IHNV isolate at a high or low dose. Brook trout mortalities induced by immersion in a bath of the RB or RA IHNV isolate at 10² plaque-forming units (pfu) per milliliter were equivalent (1 and 0%), but fish were more susceptible to infection with RA IHNV. Only the single dead fish in the RB group was infected, but 24% of the RAexposed fish were infected 1 week after exposure. At a dose of 10⁶ pfu/mL, exposure to RB IHNV resulted in a higher mortality (35%) and prevalence of infection (89% of live fish sampled at 1 week postexposure), but no infectious virus was detectable by 5 weeks after exposure. In contrast, RA IHNV exposure at a dose of 10⁴ pfu/mL resulted in only 5% mortality, and live fish killed at 1 week postexposure had a 22% prevalence of infection, but infectious virus was not detectable by week 3. Although brook trout have been previously considered to be resistant to IHNV, this study has shown that brook trout become diseased and die after exposure to a high dose of one type I IHNV isolate and can be infected after immersion exposure to even a low dose of type 1 or type 2 IHNV.     

White Sturgeon as a Potential Vector of Infectious Hematopoietic Necrosis Virus

Abstract

Cell lines from white sturgeon Acipenser transmontanus were derived from peripheral blood cells, heart, and spleen. Incubated with infectious hematopoietic necrosis virus (IHNV) for 8 d at l5°C, these cell lines produced 0.7–53.2 plaque-forming units (PFU)/cell. Waterborne exposure of larval white sturgeons (60 d posthatch) to 10⁶ PFU/mL of IHNV resulted in 10% mortality 5–6 d postinfection, with virus concentrations consistently greater than 10⁵ PFU/g. A replicate group of larval white sturgeons that were sampled at different times post-IHNV exposure had no detectable virus at 24 h, but 72% of the fish had IHNV concentrations of 10²-10⁶ PFU/g when they were examined 2–9 d postinfection. Juvenile white sturgeons (mean weight, 35 g) immersed in or injected with IHNV exhibited no mortality, and virus was only detected immediately postexposure in just 25% of the fish tested. Juvenile white sturgeons fed either virus-free rainbow trout Oncorhynchus mykiss or dead IHNV-infected rainbow trout had no viable virus in their feces. Juvenile white sturgeons fed or exposed to IHNV failed to transmit the virus to cohabiting rainbow trout fry. These results suggest that IHNV can replicate in larval white sturgeons but presumably not in juveniles or adults. Virus neutralization activity was detected in serum from adult white sturgeons (4–6 years old) cultured with rainbow trout exposed to IHNV but not in white sturgeons kept in a pathogen-free environment and fed a manufactured diet. White sturgeon serum with IHNV-neutralizing activity was used to passively immunize rainbow trout, and it provided significant (P < 0.01) protection against IHNV challenge.     

The Lethal Dose of Largemouth Bass Virus in Juvenile Largemouth Bass and the Comparative Susceptibility of Striped Bass

Abstract

Largemouth bass virus (LMBV) is an iridovirus that was isolated from wild adult largemouth bass Micropterus salmoides in the southeastern United States in 1994. Although originally isolated from moribund wild fish, its virulence to juvenile largemouth bass is uncertain. To help clarify this point, two LMBV titrations were made in juvenile largemouth bass. Titers of LMBV in fathead minnow cells were 10^4.8 and 10^5.8 tissue culture infectious doses—50% cytopathic endpoint (TCID50) per milliliter, respectively. Tenfold serial dilutions of LMBV employed in each cell culture titration, injected intraperitoneally (0.1 mL/fish) into largemouth bass produced calculated lethal dose—50% mortality endpoints (LD50s) of 282 (10^2.45) and 288 (10^2.46) infectious doses in two consecutive infectivity trials. Virus yield of assayed infected fish averaged 10^8.5 TCID50/g and 10^7.7 TCID50/g in viscera of moribund and dead fish in the two trials and 10^6.5 TCID50/g in surviving exposed fish 14 d after infection. In a second experiment, largemouth bass had 100% mortality 5 d after injection while virus immersed fish had a significantly (P ≤ 0.005) lower mortality of 17% at 14 d. Similarly treated juvenile striped bass Morone saxatilis suffered 63% mortality after injection and significantly (P ≤ 0.005) lower mortality of 10% after immersion. In a third study of 25 d, 100% of injected largemouth bass died by 5 d after injection, and all of them were virus-positive. Injected striped bass had a significantly (P ≤ 0.005) lower mortality of 24%; all three fish were virus-positive initially, two fish were virus-positive at 18 d, and none were positive at 25 d. Juvenile largemouth bass were highly susceptible to LMBV injection and striped bass were moderately susceptible, but both species were only mildly susceptible when exposed by immersion.     

Effect of Dietary Supplementation with Achyranthes aspera Seed on Larval Rohu Labeo rohita Challenged with Aeromonas hydrophila

Abstract

Larval rohu Labeo rohita were fed four different diets: three of the diets contained Achyranthes aspera (prickly chaff-flower) seeds at 0.10% (D1), 0.25% (D2), or 0.50% (D3); the fourth diet was a control diet (D4; no A. aspera supplementation). After 70 d, the rohu were injected intraperitoneally with live Aeromonas hydrophila. Mortality of fish was recorded for 7 d. In the D4 group, the first mortality was observed within 12 h of exposure, whereas in the D1–D3 treatment groups, mortality was first observed at 24 h postexposure. In the D4 group, 50% of fish died within 72 h of exposure, whereas in the D3 group, 10–15% mortality occurred between 72 and 84 h. The cumulative mortality rate was 50% for D4, 40% for D1, 35% for D2, and 15% for D3. Total tissue protein level in the larvae was higher for the D2 and D3 groups than for the other groups. Glutamic oxaloacetic transaminase, glutamate pyruvate transaminase, and thiobarbituric acid reactive substance levels were significantly lower in D3 larvae than in the other groups, whereas lysozyme and nitric oxide synthase levels were significantly higher in D3 larvae compared with the other groups. Dietary supplementation with A. aspera seeds at the 0.50% level provided protection against oxidative stress, prevented tissue damage, and enhanced disease resistance in rohu larvae.

Received December 26, 2011; accepted May 7, 2012     

Pulsed-field gel electrophoresis and multi locus sequence typing for characterizing genotype variability of Yersinia ruckeri isolated from farmed fish in France

Yersinia ruckeri is a pathogen that has an impact on aquaculture worldwide. The disease caused by this bacterial species, yersiniosis or redmouth disease, generates substantial economic losses due to the associated mortality and veterinary costs. For predicting outbreaks and improving control strategies, it is important to characterize the population structure of the bacteria. The phenotypic and genetic homogeneities described previously indicate a clonal population structure as observed in other fish bacteria. In this study, the pulsed-field gel electrophoresis (PFGE) and multi locus sequence typing (MLST) methods were used to describe a population of isolates from outbreaks on French fish farms. For the PFGE analysis, two enzymes (NotI and AscI) were used separately and together. Results from combining the enzymes showed the great homogeneity of the outbreak population with a similarity > 80.0% but a high variability within the cluster (cut-off value = 80.0%) with a total of 43 pulsotypes described and an index of diversity = 0.93. The dominant pulsotypes described with NotI (PtN4 and PtN7) have already been described in other European countries (Finland, Germany, Denmark, Spain and Italy). The MLST approach showed two dominant sequence types (ST31 and ST36), an epidemic structure of the French Y. ruckeri population and a preferentially clonal evolution for rainbow trout isolates. Our results point to multiple types of selection pressure on the Y. ruckeri population attributable to geographical origin, ecological niche specialization and movements of farmed fish.     

Development and evaluation of a multiplex PCR assay for simultaneous detection of Flavobacterium psychrophilum, Yersinia ruckeri and Aeromonas salmonicida subsp. salmonicida in culture fisheries

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.     

Influence of Infection with Renibacterium salmoninarum on Susceptibility of Juvenile Spring Chinook Salmon to Gas Bubble Trauma

Abstract

During experiments in our laboratory to assess the progression and severity of gas bubble trauma (GBT) in juvenile spring chinook salmon Oncorhynchus tshawytscha, we had the opportunity to assess the influence of Renibacterium salmoninarum (Rs), the causative agent of bacterial kidney disease, on the susceptibility of salmon to GBT. We exposed fish with an established infection of Rs to 120% total dissolved gas (TDG) for 96 h and monitored severity of GBT signs in the fins and gills, Rs infection level in kidneys by using an enzyme-linked immunosorbent assay (ELISA), and mortality. Mortality occurred rapidly after exposure to 120% TDG, with a LT20 (time necessary to kill 20% of the population) of about 37 h, which is at a minimum about 16% earlier than other bioassays we have conducted using fish that had no apparent signs of disease. Fish that died early (from 31 to 36 h and from 49 to 52 h) had significantly higher infection levels (mean ± SE ELISA absorbance = 1.532 ± 0.108) than fish that survived for 96 h (mean ± SE ELISA absorbance = 0.828 ± 0.137). Fish that died early also had a significantly greater number of gill filaments occluded with bubbles than those that survived 96 h. Conversely, fish that survived for 96 h had a significantly higher median fin severity ranking than those that died early. Our results indicate that fish with moderate to high levels of Rs infection are more vulnerable to the effects of dissolved gas supersaturation (DGS) and die sooner than fish with lower levels of Rs infection. However, there is a substantial amount of individual variation in susceptibility to the apparent cumulative effects of DGS and Rs infection. Collectively, our findings have important implications to programs designed to monitor the prevalence and severity of GBT in juvenile salmonids in areas like the Columbia River basin, and perhaps elsewhere.     

Establishment of inflammatory model induced by Pseudorabies virus infection in mice

BackgroundPseudorabies virus (PRV) infection leads to high mortality in swine. Despite extensive efforts, effective treatments against PRV infection are limited. Furthermore, the inflammatory response induced by PRV strain GXLB-2013 is unclear.ObjectivesOur study aimed to investigate the inflammatory response induced by PRV strain GXLB-2013, establish an inflammation model to elucidate the pathogenesis of PRV infection further, and develop effective drugs against PRV infection.MethodsKunming mice were infected intramuscularly with medium, LPS, and different doses of PRV-GXLB-2013. Viral spread and histopathological damage to brain, spleen, and lung were determined at 7 days post-infection (dpi). Immune organ indices, levels of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines, as well as levels of activity of COX-2 and iNOS were determined at 4, 7, and 14 dpi.ResultsAt 10⁵–10⁶ TCID₅₀ PRV produced obviously neurological symptoms and 100% mortality in mice. Viral antigens were detectable in kidney, heart, lung, liver, spleen, and brain. In addition, inflammatory injuries were apparent in brain, spleen, and lung of PRV-infected mice. Moreover, PRV induced increases in immune organ indices, ROS and NO levels, activity of COX-2 and iNOS, and the content of key pro-inflammatory cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor-α, interferon-γ and MCP-1. Among the tested doses, 10² TCID₅₀ of PRV produced a significant inflammatory mediator increase.ConclusionsAn inflammatory model induced by PRV infection was established in mice, and 10² TCID₅₀ PRV was considered as the best concentration for the establishment of the model.     

Competitive exclusion of Yersinia enterocolitica biotype 4, serotype O:3 by Yersinia enterocolitica biotype 1A,serotype O:6,30 in tissue culture and in pigs

AIMS: To study the adhesion properties of a biotype 4, serotype O:3 (human pathogenic) strain of Yersinia enterocolitica and to determine if adhesion in vitro and colonisation in vivo can be prevented by competition with a biotype 1A, serotype O:6,30 (non-pathogenic) strain. To study interaction between Y. enterocolitica biotype 4, serotype O:3 and cultured epithelial cells using the synthetic tripeptide arginine-glycine-aspartic acid (RGD).

METHODS: The human intestinal epithelial (HEp-2) cell line was used for in vitro studies. Inocula of Y. enterocolitica biotype 4, serotype O:3 radiolabelled using tritium were incubated with HEp-2 cells and RGD tripeptide, or with Y. enterocolitica biotype 1A, serotype O:6,30 sequentially or concurrently, then washed and lysed, and radioactivity measured to determine the effect of RGD on adhesion, and competitive exclusion of pathogenic by non-pathogenic bacteria. For in vivo studies, two groups of 5-week-old piglets (n=5/group) were sequentially inoculated orally with 5x10⁹ colony forming units (cfu) of either a non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica followed by a pathogenic biotype 4, serotype O:3 strain, or vice versa. Pigs were monitored for carriage of strains using bacterial culture and a multiplex polymerase chain reaction (PCR).

RESULTS: The RGD tripeptide significantly inhibited adherence of the pathogenic Y. enterocolitica strain to cultured epithelial cells, suggesting that adhesion involved the RGD tripeptide sequence. The non-pathogenic biotype 1A, serotype O:6,30 strain of Y. enterocolitica prevented adhesion of the pathogenic strain to cells in vitro when allowed to adhere first. Pathogenic Y. enterocolitica was consistently isolated from rectal swabs from 80-100% of pigs on all sampling occasions but not from oral swabs after 14 days in pigs first inoculated with the non- pathogenic strain or at 26 days in pigs first inoculated with the pathogenic strain.

CONCLUSIONS: A non-pathogenic strain of Y. enterocolitica reduced adhesion of a human pathogenic strain in vitro but not in vivo.     

Effects of Formalin and Chloramine-T Treatments on Oxygen Consumption of Juvenile Salmonids

Abstract

The effects of formalin and chloramine-T on oxygen consumption of juvenile brook trout Salvelinus fontinalis at low water temperature were studied with a flow-through respirometer. No changes were found in the oxygen consumption of these fish after exposure to formalin at 200 and 400 μL/L or to chloramine-T at 10 μL/L during 1-h flow-through treatments. Additionally, there was no evidence of chemical oxygen demand exerted by formalin at dose levels up to 1,600 μL/L or by chloramine-T at I0 μL/L. When juvenile rainbow trout Oncorhynchus mykiss were treated with formalin at 200 μL/L for 1 h in an aerated tank simulating a static bath treatment, sham-treated control tanks had a significantly greater maximum decline in dissolved oxygen than formal in-treated tanks. These studies suggest that posttreatment mortalities that occur after low-temperature bath treatments with these chemicals are not directly related to oxygen consumption.     

Isolation and Pathogenicity of Streptococcus iniae in Cultured Red Hybrid Tilapia in Malaysia

Abstract

This study describes the isolation and pathogenicity of Streptococcus iniae in cultured red hybrid tilapia (Nile Tilapia Oreochromis niloticus × Mozambique Tilapia O. mossambicus) in Malaysia. The isolated gram-positive S. iniae appeared punctiform, transparently white, catalase and oxidase negative and produced complete β-hemolysis on blood agar, while a PCR assay resulted in the amplification of the 16 S rRNA gene and lactate oxidase encoded genes. The isolate was sensitive to tetracycline, vancomycin, and bacitracin but was resistant to streptomycin, ampicillin, penicillin, and erythromycin. Pathogenicity trials conducted in local red hybrid tilapia (mean ± SE = 20.00 ± 0.45 g) showed 90.0, 96.7, and 100.0% mortality within 14 d postinfection following intraperitoneal exposure to 10⁴, 10⁶, and 10⁸ CFU/mL of the pathogen, respectively. The clinical signs included erratic swimming, lethargy, and inappetance at 6 h postinfection, while mortality was recorded at less than 24 h postinfection in all infected groups. The LD_{50-336 h} of S. iniae against the red hybrid tilapia was 10² CFU/mL. The post mortem examinations revealed congested livers, kidneys, and spleens of the infected fish. This is the first report of S. iniae experimental infection in cultured red hybrid tilapia in Malaysia.

Received January 20, 2017; accepted July 16, 2017 Published online October 11, 2017     

Host factors associated with the detection of Aeromonas salmonicida and Yersinia ruckeri in Ontario, Canada government fish hatcheries

This paper presents an epidemiological investigation of Ontario Ministry of Natural Resources Fish Health Laboratory data from 1981 to 1997, to determine whether fish species and age were associated with lot-level detection of Aeromonas salmonicida and Yersinia ruckeri in hatchery fish. In stepwise logistic regression, the species brook trout and back-cross (lake trout crossed with the hybrid “splake”) were more likely to test A. salmonicida-positive compared to all other species reared in the hatcheries. Similarly, the species brook trout was significantly more likely to test Y. ruckeri-positive compared to all other species. For both pathogens, the 1–5-month age group was associated significantly with detection. These findings suggest that purposive sampling of higher-risk fish lots could increase the likelihood of detecting both study pathogens.     

Susceptibility of Fish and Turtles to Three Ranaviruses Isolated from Different Ectothermic Vertebrate Classes

Abstract

Ranaviruses have been associated with mortality of lower vertebrates around the world. Frog virus 3 (FV3)-like ranaviruses have been isolated from different ectothermic vertebrate classes; however, few studies have demonstrated whether this pathogen can be transmitted among classes. Using FV3-like ranaviruses isolated from the American bullfrog Lithobates catesbeianus, eastern box turtle Terrapene carolina carolina, and Pallid Sturgeon Scaphirhynchus albus, we tested for the occurrence of interclass transmission (i.e., infection) and host susceptibility (i.e., percent mortality) for five juvenile fish and three juvenile turtle species exposed to each of these isolates. Exposure was administered via water bath (10³ PFU/mL) for 3 d and survival was monitored for 28 d. Florida softshell turtles Apalone ferox experienced no mortality, but 10% and 20% of individuals became infected by the turtle and fish isolate, respectively. Similarly, 5% of Mississippi map turtles Graptemys pseudogeographica kohni were subclinically infected with the turtle isolate at the end of the experiment. Channel Catfish Ictalurus punctatus experienced 5% mortality when exposed to the turtle isolate, while Western Mosquitofish Gambusia affinis experienced 10% mortality when exposed to the turtle and amphibian isolates and 5% mortality when exposed to the fish isolate. Our results demonstrated that interclass transmission of FV3-like ranaviruses is possible. Although substantial mortality did not occur in our experiments, the occurrence of low mortality and subclinical infections suggest that fish and aquatic turtles may function as reservoirs for FV3-like ranaviruses. Additionally, our study is the first to report transmission of FV3-like ranaviruses between fish and chelonians.

Received October 22, 2013; accepted January 8, 2014.