Negative Control Fish Research Articles

Susceptibility of Pallid Sturgeon to viral hemorrhagic septicemia virus genotype IVb.

Viral hemorrhagic septicemia virus (VHSV) is an aquatic rhabdovirus causing severe disease in freshwater and saltwater fish species. The susceptibility of endangered Pallid Sturgeon Scaphirhynchus albus to VHSV genotype IVb (VHSV-IVb) infection was investigated. An in vitro assessment using two Pallid Sturgeon cell lines derived from skin and spleen tissue and in vivo evaluation of juvenile Pallid Sturgeon after exposure to VHSV-IVb were performed. Plaque assay and RT-PCR results confirmed VHSV-IVb replication in Pallid Sturgeon cell lines. Sturgeon were also susceptible to VHSV-IVb infection after immersion and injection exposures during laboratory experiments. However, after widespread mortality occurred in all treatment groups, including negative control fish, it was determined that the Pallid Sturgeon stock fish were infected with Missouri River sturgeon iridovirus (MRSIV) prior to experimental challenge. Nevertheless, mortalities were equal or higher among VHSV-exposed fish than among negative controls (MRSIV infected), and histopathological assessments indicated reduced hematopoietic cells in spleen and kidney tissues and hemorrhage in the gastrointestinal organs only in fish from the VHSV treatment. These results indicate that Pallid Sturgeon is a susceptible host for VHSV-IVb, but the degree of pathogenicity was confounded by the underlying MRSIV infection. Research comparing susceptibility of specific pathogen-free and MRSIV-infected fish to VHSV-IVb is needed to accurately assess the vulnerability of Pallid Sturgeon to VHSV-IVb.

Immune response of large yellow croaker Larimichthys crocea towards a recombinant vaccine candidate targeting the parasitic ciliate Cryptocaryon irritans.

Cryptocaryon irritans, a parasitic ciliate, pose a major threat to marine teleost fish aquaculture. So far, no effective and safe control method is available. In this study, the protective efficacy of a recombinant truncated surface antigen of C. irritans (rCiSA32.6t) for large yellow croaker (Larimichthys crocea) against the parasite challenge with a sub-lethal dose of the infective theronts was evaluated by comparing the relative percent survivals (RPS), the specific antibody titers in sera, and the expression levels of the immune-related genes among the negative or adjuvant control fish, fish intraperitoneally immunized with rCiSA32.6t. The results showed that a RPS of 50.1% in rCiSA32.6t-immunized fish was achieved in comparison to negative control fish against C. irritans. A significant increase was noted in the antigen-specific immunoglobulin M (IgM) and immunoglobulin T (IgT) antibody levels in the sera of the rCiSA32.6t-vaccinated fish. Compared to the negative control fish, quantitative real-time PCR analysis indicated that the interleukin-1beta, IgT, and IgM heavy chain mRNA level in the fish head kidney, spleen, gill, and skin tissue were upregulated post-rCiSA32.6t immunization. This study indicates that the rCiSA32.6t can provide a high level of immune protection against C. irritans infection in grouper and is therefore pursued as a candidate C. irritans vaccine.

Gut Microbiome as a Potential Biomarker in Fish: Dietary Exposure to Petroleum Hydrocarbons and Metals, Metabolic Functions and Cytokine Expression in Juvenile Lates calcarifer

The gut microbiome of fish contains core taxa whose relative abundances are modulated in response to diet, environmental factors, and exposure to toxicogenic chemicals, influencing the health of the host fish. Recent advances in genomics and metabolomics have suggested the potential of microbiome analysis as a biomarker for exposure to toxicogenic compounds. In this 35-day laboratory study, 16S RNA sequencing and multivariate analysis were used to explore changes in the gut microbiome of juvenile Lates calcarifer exposed to dietary sub-lethal doses of three metals: vanadium (20 mg/kg), nickel (480 mg/kg), and iron (470 mg/kg), and to two oils: bunker C heavy fuel oil (HFO) (1% w/w) and Montara, a typical Australian medium crude oil (ACO) (1% w/w). Diversity of the gut microbiome was significantly reduced compared to negative controls in fish exposed to metals, but not petroleum hydrocarbons. The core taxa in the microbiome of negative control fish comprised phyla Proteobacteria (62%), Firmicutes (7%), Planctomycetes (3%), Actinobacteria (2%), Bacteroidetes (1%), and others (25%). Differences in the relative abundances of bacterial phyla of metal-exposed fish were pronounced, with the microbiome of Ni-, V-, and Fe-exposed fish dominated by Proteobacteria (81%), Firmicutes (68%), and Bacteroidetes (48%), respectively. The genus Photobacterium was enriched proportionally to the concentration of polycyclic aromatic hydrocarbons (PAHs) in oil-exposed fish. The probiotic lactic acid bacterium Lactobacillus was significantly reduced in the microbiota of fish exposed to metals. Transcription of cytokines IL-1, IL-10, and TNF-a was significantly upregulated in fish exposed to metals but unchanged in oil-exposed fish compared to negative controls. However, IL-7 was significantly downregulated in fish exposed to V, Ni, Fe, and HFOs. Fish gut microbiome exhibits distinctive changes in response to specific toxicants and shows potential for use as biomarkers of exposure to V, Ni, Fe, and to PAHs present in crude oil.

An inactivated vaccine against infectious pancreatic necrosis virus in rainbow trout (Oncorhynchus mykiss)

Infectious pancreatic necrosis virus (IPNV), belonging to the genus Aquabirnavirus within the family Birnaviridae, causes huge economic loss to the global salmonid industry every year. Recently, outbreaks of disease caused by genogroup I IPNV were found in many rainbow trout (Oncorhynchus mykiss) farms worldwide. An inactivated vaccine was prepared using a genogroup I IPNV isolate with an optimized procedure as incubation with β-propanolactone (BPL) at the final concentration of 0.5% at room temperature for 48 h. The inactivated vaccine was used to immunize rainbow trout, and the protection efficiency was evaluated by viral loads determination, immune-related genes quantification, and neutralizing antibody tests. The viral loads in immunized rainbow trout were significantly decreased and the strongest antiviral effect was observed on 30 days post-immunization (d.p.i). The expression of innate immune-related genes IFN-1, and Mx-1 genes were significantly up-regulated on 3, 7, and 15 d.p.i (p < 0.05), and adaptive immune-related genes CD4, CD8, and IgM genes were significantly up-regulated on 15 and 30 d.p.i (p < 0.05). Neutralizing antibodies were firstly detected on 30 d.p.i and the highest titer was observed on 45 d.p.i, which began to decrease on 60 d.p.i, but was still significantly higher than that in negative control fish. The results indicated that the vaccine prepared in this study could stimulate the non-specific and specific immune response and provide significant immune protection to the vaccinated rainbow trout.

The Effectiveness of Turmeric Enriched Pellets in Curing the Gill of Clarias batrachus Infected with Aeromonas hydrophila

Curcumin is an antibiotic agent extracted from Curcuma longa. To understand the effectiveness of curcumin in curing the gill of Clarias batrachus infected with Aeromonas hydrophila, a study was conducted from August to November 2020. There were 3 treatments applied, namely turmeric enriched pellets 0.7g/kg (P1); 0.9g/kg (P2) and 1.1g/kg (P3). Prior to the treatment, the fish was infected with A. hydrophila (0.1 m of A. hydrophila culture, 1.0x108cells/ml). The infected fish was kept for 6 days until the clinical signs of MAS disease occurred. By the 7th day, the fish was feed with turmeric enriched pellets. For negative control (Cn) the fish was not infected with A. hydrophila nor treated with turmeric, while the positive control (Cp) was infected, but no turmeric treatment. The fish was reared for 30 days and by the end of the experiment, the gill was removed and processed for histological study (5L sliced and HE stained). Result indicated that the gill of the negative control fish was normal, while that of the positive control shown abnormalities such as hemorrhages, necrotic cells, fused lamellae and enlarged cartilaginous cells. The turmeric treated fish shown less damage in the gill. The best results were obtained in C3, as the gill structure was almost normal with slightly hemorrhage. In the C1 and C2 treated fishes, the gill shown light abnormality such as fused lamella and hemorrhage. Data obtained shown that the consumption of turmeric enriched pellet was effective to cure the A. hydrophyla infected gill.

Application of the FTA elute card coupled with visual colorimetric loop-mediated isothermal amplification for the rapid diagnosis of Streptococcus agalactiae in farmed tilapia (Oreochromis niloticus).

A method combining the FTA Elute card and visual colorimetric loop-mediated isothermal amplification (FTA-e/LAMP) was tested to diagnose Streptococcus agalactiae infections in vitro and in vivo. FTA-e/LAMP consists of two main steps: first, the FTA card is used to extract DNA and then a colorimetric loop-mediated isothermal amplification (LAMP) reaction is carried out on the extracted DNA. In vitro sensitivity was 1.9x102 CFU/mL, and regarding specificity, all nine S.agalactiae strains tested positive. All Streptococcus spp. tested negative, except for S.dysgalactiae, thereby indicating the need for another set of primers to distinguish this species from S.agalactiae. To diagnose S.agalactiae infections using FTA-e/LAMP in vivo, two experimental trials on juvenile Oreochromis niloticus infected with bovine or piscine strains were carried out. Sensitivity in symptomatic fish was 100%, and 50.7% of fish without signs were positive. All negative control fish tested negative (n=28). No bacteria were detected after 16days post-infection (dpi). Accuracy during the first week (1-7dpi) was 89% and decreased to 44% thereafter (10-22dpi). FTA-e/LAMP results suggest that this method is a promising tool for early and fast diagnosis of S.agalactiae on tilapia farms.

Effects of diet supplementation of soya-saponins, isoflavones and phytosterols on Atlantic salmon (Salmo salar, L) fry fed from start-feeding

A 14-week trial was conducted to investigate the effects of antinutritional factors (ANFs) commonly present in soybean ingredients, singly and in combination, on Atlantic salmon (Salmo salar L.) fed from start-feeding. The experimental diets consisted of a negative control fish meal diet (FM), and a positive control diet with 167 g kg−1 soybean meal inclusion (SBM) and four diets based on the FM diet supplemented with 2 g kg−1 soya-saponins (SAP), 1.5 g kg−1 isoflavones (IFL), 0.3 g kg−1 phytosterols (PHS) or a mixture of these (MIX). Fish fed the SAP diet showed significantly higher growth performance than those fed FM, while the IFL treatment significantly decreased growth performance of salmon fry. Fish fed the IFL diet had significantly lower maltase activity and higher trypsin activity in proximal intestine than fish fed the FM diet. Histological differences were observed in the liver of fish fed the IFL diet, characterized by reduced size of the hepatocytes. Fish fed the PHS and IFL diets showed the highest frequencies of skeletal deformities among the six treatments. In conclusion, the results indicate that purified isoflavones may negatively affect growth performance, intestinal function, liver metabolism and bone formation of salmon fry.

Evaluation of guppy (Poecilia reticulata Peters) immunization against Tetrahymena sp. by enzyme-linked immunosorbent assay (ELISA)

Analysis of the effectiveness of guppy (Poecilia reticulata Peters) immunization based on measurements of antibody (Ab) titers suffers from a shortage of reagents that can detect guppy antibodies (Abs). To overcome this problem, we immunized mice with different preparations of guppy immunoglobulins (Igs) and used the mouse antisera to develop a quantitative enzyme-linked immunosorbent assay (ELISA). The most efficient immunogen for mouse immunization was guppy Igs adsorbed on protein A/G beads. Antisera from mice boosted with this immunoglobulin (Ig) preparation were highly specific and contained high Ab titers. They immunoreacted in a Western blot with Ig heavy and light chains from guppy serum, and Ig heavy chain from guppy whole-body homogenate. The mouse anti-guppy Ig was applied in an ELISA aimed at comparing the efficiency of different routes of guppy immunization against Tetrahymena: (i) anal intubation with sonicated Tetrahymena (40,000 Tetrahymena/fish in a total volume of 10μL) mixed with domperidon, deoxycholic acid and free amino acids (valine, leucine, isoleucine, phenylalanine and tryptophan), or (ii) intraperitoneal (i.p.) injection of sonicated Tetrahymena in complete Freund's adjuvant (15,000 Tetrahymena/fish in total a volume of 20μL). Negative control fish were anally intubated with the intubation mixture without Tetrahymena, or untreated. ELISA measurement of anti-Tetrahymena Ab titer revealed a significantly higher level of Abs in i.p.-immunized guppies, compared to the anally intubated and control fish. In addition, the efficiency of immunization was tested by monitoring guppy mortality following (i) i.p. challenge with Tetrahymena (900 Tetrahymena/fish) or (ii) cold stress followed by immersion in water containing 10,000 Tetrahymena/mL. Fish mortality on day 14 post-Tetrahymena infection by i.p. injection exceeded 50% in the control and anally intubated fish, compared to 31% in i.p.-immunized fish. Immunization did not protect from pathogen challenge by immersion. The results suggest a direct correlation between the anti-Tetrahymena Ab response and fish resistance to i.p.-injected Tetrahymena, but not to infection by immersion preceded by cold stress.

Induction of micronucleus of Oreochromis niloticus exposed to waters from the Cubatão do Sul River, southern Brazil

In an effort to characterize the pollution of surface waters by potentially genotoxic agents, this study aimed at assessing the frequency of micronucleated (MN) erythrocytes of the fish species, Oreochromis niloticus, from the Cubatão do Sul River. This river is the source of drinking water for the region of Florianópolis, capital of Santa Catarina State, Brazil. Negative control fish showed low frequency of MN, ranging between 0.49‰ and 0.90‰. Positive control (potassium dichromate 2.5mg/L) organisms showed high MN frequency (16.82–17.25‰). The MN frequency increased along the river (Site 1 – 1.24‰ winter 2011; Site 4 – 9.76‰ summer 2011).Based on the observation of elevated MN erythrocytes frequency in O. niloticus exposed to water samples from along the river course, we conclude that the complex environmental mixtures of water from the Cubatão do Sul River have genotoxic potential. This genotoxicity most likely originated from agricultural runoff and domestic effluents released without treatment, based on the evidence from literature data and a survey in the region. This study provides a scientific basis for future studies regarding the genotoxicity of complex environmental mixtures in natural environments.

Construction of a DNA Vaccine Using Glycoprotein Gene and Its Expression Towards Increasing Survival Rate of KHV-Infected Common Carp (Cyprinus carpio)

Deoxyribonucleic acid (DNA) vaccine has recently been developed as an alternative vaccine against virus infection.This study was the first step of DNA vaccine development to protect cyprinids including common carp (Cyprinuscarpio) and fancy koi (Cyprinus carpio) from KHV (koi herpesvirus) infection in Indonesia. One of KHV glycoproteingenes, i.e. glycoprotein (GP) was ligated with Japanese medaka (Oryzias latipes) â-actin promoter to generatepAct/GP as a DNA vaccine. Fourty fish in body weight of 10-15 g/fish were individually injected by pAct/GP intomuscle in different dosage of 2.5 μg, 7.5 μg and 12.5 μg/100 μl phosphate buffer saline. Total RNA was extractedfrom the 12.5 μg of pAct/GP-injected fish muscle at 24, 48 and 67 hours post-injection to analyze GP expression byRT-PCR method. Potential of pAct/GP as DNA vaccine was examined by injecting KHV into the 30-days-vaccinatedfish. Both of possitive and negative control fish group were not vaccinated. Possitive control fish group wereinjected with KHV, but negative control fish group were not. KHV-challenged fish were reared for 1 month, and thedeath fish were calculated daily. Result of RT-PCR analysis showed that GP gene expression were detected at 3 dpost-injection. Expression of GP in the vaccinated fish groups helped to improve their survival rate after challengedby KHV. All of fish without DNA vaccination had dead 17 days after KHV injection. The results demonstrated thatpAct/GP had high potency to be used as a DNA vaccine against KHV infection in cyprinids.

Impact of Thiamine Deficiency on T‐cell Dependent and T‐cell Independent Antibody Production in Lake Trout

Lake trout Salvelinus namaycush on thiamine-replete and thiamine-depleted diets were evaluated for the effects of thiamine status on in vivo responses to the T-dependent antigen trinitophenol (TNP)-keyhole limpet hemocyanin (TNP-KLH), the T-independent antigen trinitrophenol-lipolysaccaharide (TNP-LPS), or Dulbecco's phosphate-buffered saline (DPBS; negative control fish). Plasma antibody concentrations were evaluated for possible differences in total anti-TNP activity as well as differences in response kinetics. Associations between anti-TNP activity and muscle and liver thiamine concentrations as well as ratios of muscle-to-liver thiamine to anti-TNP activity were also examined. Thiamine-depleted lake trout that were injected with TNP-LPS exhibited significantly more anti-TNP activity than thiamine-replete fish. The depleted fish injected with TNP-LPS also exhibited significantly different response kinetics relative to thiamine-replete lake trout. No differences in activity or kinetics were observed between the thiamine-replete and -depleted fish injected with TNP-KLH or in the DPBS negative controls. Anti-TNP activity in thiamine-depleted lake trout injected with TNP-KLH was positively associated with muscle thiamine pyrophosphate (thiamine diphosphate; TPP) concentration. A negative association was observed between the ratio of muscle-to-liver TPP and T-independent responses. No significant associations between anti-TNP activity and tissue thiamine concentration were observed in the thiamine-replete fish. We demonstrated that thiamine deficiency leads to alterations in both T-dependent and T-independent immune responses in lake trout.

Development of neutralizing antibody responses in muskellunge, Esox masquinongy (Mitchill), experimentally exposed to viral haemorrhagic septicaemia virus (genotype IVb)

A complement-dependent 50% plaque neutralization test was used to assess the neutralizing antibody response in sera of muskellunge, Esox masquinongy, experimentally infected with viral haemorrhagic septicaemia virus (VHSV, genotype IVb) by immersion. Groups of muskellunge were challenged with varying concentrations of VHSV: Group 1 with 10(2) plaque-forming units (pfu) mL(-1) , Group 2 with 4 × 10(3) pfu mL(-1) , Group 3 with 10(5) pfu mL(-1) and Group 4 with 0 pfu mL(-1) . The fish were held at a temperature of 11 ± 1 °C and were sampled over a 20-week period. Neutralizing antibodies were not detected in sera of any of the negative control fish throughout the study. Low neutralizing titres were detected in Groups 1-3 by 6 days post-infection (p.i.). Neutralizing titres of ≥80 [corrected]. were not detected again until 3, 4 and 7 weeks p.i. for Groups 2, 3 and 1, respectively, with peak titres for those groups occurring 16, 11 and 17 weeks p.i., respectively. VHSV was detected in serum for up to 11 weeks p.i. Results of this study show that survivors can be detected by a serological technique, despite being virus negative. This may benefit the investigation of VHSV IVb distribution in the Great Lakes and the study of host immune responses to this emerging sublineage.

Effect of β-glucan on activity of antioxidant enzymes and Mx gene expression in virus infected grass carp

The effects of β-glucan, an immunostimulatory agent, on the superoxide dismutase (SOD) and catalase (CAT) activities of erythrocytes and Mx gene expression were studied from grass carp that were challenged with grass carp hemorrhage virus (GCHV). The SOD and CAT activities in erythrocytes and Mx gene expression in spleen from the fish were detected by spectrophotometry and RT-PCR, respectively. Negative control fish were injected with PBS; positive control groups were injected with either β-glucan or GCHV only; and the experimental groups were pre-injected with β-glucan 15 days prior to injection with GCHV. The results show that the SOD and CAT activities were higher in fish injected with β-glucan for 15 days than the negative control group injected with PBS. The SOD and CAT activities significantly decreased when the fish were challenged with GCHV, but it was higher in the group pre-treated with β-glucan than in infected fish not pre-treated, 15 days after GCHV infection. Mx gene expression levels increased during the early stages (at 12 h and 36 h) of GCHV infection, and it remained at higher levels from the 6th till the 10th day in the β-glucan pre-treated group, but it was falling from the 6th day in the β-glucan untreated group. The GCHV-infected group pre-treated with β-glucan had a higher survival rate (60%) than the group not pre-treated with β-glucan (20%), suggesting that β-glucan possesses or enhances anti-viral responses.

Effect of isoeugenol sedation on plasma cortisol, glucose, and lactate dynamics in channel catfish Ictalurus punctatus exposed to three stressors

The effect of isoeugenol sedation (2.5 mg l −1) on plasma cortisol, glucose, and lactate dynamics in channel catfish ( Ictalurus punctatus), relative to two controls, was examined during confinement, exposure to high unionized ammonia, and acute oxygen depletion. The positive control (PC) for each treatment was no sedation, and the negative control (NC) was sedation with metomidate hydrochloride (1.5 mg l −1). Isoeugenol sedated catfish had 60% lower ( P<0.05) plasma cortisol levels than PC fish after 15-min of confinement. No differences ( P>0.05) in plasma levels of glucose or lactate were observed between treatments during 45-min of confinement. Fish exposed to high ammonia for 24-h had elevated ( P<0.05) cortisol levels in PC and isoeugenol treatment groups. Sedation with isoeugenol during exposure to high ammonia had no effect on plasma glucose or lactate levels relative to PC fish. Levels of plasma cortisol, glucose, and lactate all increased significantly ( P<0.05) in PC fish following acute oxygen depletion for 30 min. Plasma glucose levels were similar ( P>0.05) between isoeugenol and control treated fish throughout oxygen stress and recovery. Sedation with isoeugenol significantly suppressed the resulting plasma cortisol and lactate response by 74% and 46%, respectively, compared to PC fish. Plasma cortisol levels in NC fish were below detection limits for all stress treatments. As a sedative, isoeugenol had suppressive effects on channel catfish plasma cortisol levels during confinement and low oxygen conditions, and reduced the plasma lactate response to acute oxygen depletion.

Surface immobilization antigen of the parasitic ciliate Ichthyophthirius multifiliis elicits protective immunity in channel catfish (Ictalurus punctatus).

Channel catfish (Ictalurus punctatus) that survive infection with the parasitic ciliate Ichthyophthirius multifiliis acquire immunity to subsequent challenge and produce specific antibodies in serum that immobilize the parasite in vitro. Cellular surface protein antigens targeted by these antibodies are referred to as immobilization antigens (i-antigens). By using an immobilizing mouse monoclonal antibody as a ligand, the i-antigen of I. multifiliis isolate G5 was purified to homogeneity by immunoaffinity chromatography, and its immunogenicity was confirmed by inoculating rabbit and channel catfish to produce immobilizing antisera. To test the purified i-antigen as a subunit vaccine, channel catfish fingerlings were injected intraperitoneally (i.p.) with purified i-antigen at a dose of 10 microg/fish in complete Freund's adjuvant on day 1, followed by a second i.p. injection of the same amount of i-antigen in incomplete Freund's adjuvant on day 15. Negative control fish were immunized similarly with either bovine serum albumin (BSA) or an immobilization-irrelevant I. multifiliis protein. On day 84, the fish were challenged with live I. multifiliis G5 theronts at a dose of 15,000 cells per fish. Seventy-two percent of the fish immunized with i-antigen survived the challenge. All negative control fish died within 16 days of exposure. There was a significant difference in the median days to death between the negative control fish injected with BSA and the fish that died following vaccination with i-antigen. Fish injected with i-antigen developed high immobilizing antibody titers in serum. This is the first demonstration of a direct role for i-antigens in the elicitation of protective immunity, suggesting that these proteins by themselves serve as effective subunit vaccines against I. multifiliis.

Temperature effect on nervous necrosis virus infection in grouper cell line and in grouper larvae

This preliminary study elucidates the in vitro and in vivo effects of temperature on grouper nervous necrosis virus (GNNV) infection. A novel continuous cell line derived from the fin tissue of a grouper ( Epinephelus coioides, Hamilton), named as GF-1 cell line, was used. Cytopathic effect was observed in GNNV-infected GF-1 cells incubated at 24–32°C after viral adsorption, but not at 20°C or 37°C even though the viral adsorption temperature was 28°C. Viral protein could be detected in the pellets of GNNV-infected GF-1 cells cultured at 20–32°C, but not at 37°C. In a challenge test, GNNV-challenged larvae which were maintained at a constant 28°C began to die 1 day post challenge (p.c.) with a death rate of 80%. Mortality reached 100% by 50 h p.c., while the mortality of negative control fish was only 5%. The cumulative mortality of GNNV-challenged larvae at ambient temperature, i.e. 28°C at noon and 24°C at midnight, was 10% 1 day p.c., and increased to 100% by 80 h p.c. Based on the results, we concluded that temperature plays an important role in GNNV infection and pathogenicity.

Relative virulence of three isolates of Piscirickettsia salmonis for coho salmon Oncorhynchus kisutch.

Piscirickettsia salmonis was first recognized as the cause of mortality among pen-reared coho salmon Oncorhynchus kisutch in Chile. Since the initial isolation of this intracellular Gram-negative bacterium in 1989, similar organisms have been described from several areas of the world, but the associated outbreaks were not reported to be as serious as those that occurred in Chile. To determine if this was due to differences in virulence among isolates of P. salmonis, we conducted an experiment comparing isolates from Chile, British Columbia, Canada, and Norway (LF-89, ATL-4-91 and NOR-92, respectively). For each of the isolates, 3 replicates of 30 coho salmon were injected intraperitoneally with each of 3 concentrations of the bacterium. Negative control fish were injected with MEM-10. Mortalities were collected daily for 41 d post-injection. Piscirickettsiosis was observed in fish injected with each of the 3 isolates, and for each isolate, cumulative mortality was directly related to the concentration of bacterial cells administered. The LF-89 isolate was the most virulent, with losses reaching 97% in the 3 replicates injected with 10(5.0) TCID50, 91% in the replicates injected with 10(4.0) TCID50, and 57% in the fish injected with 10(3.0) TCID50. The ATL-4-91 isolate caused losses of 92% in the 3 replicates injected with 10(5.0) TCID50, 76% in the fish injected with 10(4.0) TCID50, and 32% in those injected with 10(3.0) TCID50. The NOR-92 isolate was the least virulent, causing 41% mortality in the replicates injected with 10(4.6) TCID50. At 41 d post-injection, 6% of the fish injected with 10(3.6) TCID50 NOR-92 had died. Mortality was only 2% in the fish injected with 10(2.6) TCID50 NOR-92, which was the same as the negative control group. Because the group injected with the highest concentration (10(4.6) TCID50) of NOR-92 was still experiencing mortality at 41 d, it was held for an additional 46 d. At 87 d post-injection, the cumulative mortality in this group had reached 70%. These differences in virulence among the isolates were statistically significant (p < 0.0001), and are important for the management of affected stocks of fish.