Cold Water Disease Research Articles

Penicillin-G: Efficacy against <i>Flavobacterium psychrophilum</i> and evaluation of lethal dose limits for rainbow trout

The effect of penicillin-G on Flavobacterium psychrophilum, the cause of bacterial coldwater disease, was evaluated in 15 min and overnight exposures. Separate tests evaluated the effect of increasing doses of penicillin-G to rainbow trout Oncorhynchus mykiss eggs, fingerlings, and adults. Rainbow trout eggs were exposed for 1 h to penicillin doses of 0, 250, 500, 1,000, 10,000, 50,000, or 100,000 IU/mL. Mean percent hatch ranged from 64.6% to 75.1%, and did not significantly differ among the treatments. Fingerlings were injected intraperitoneally (i.p.) with 200 μL of 0, 1, 10, 100, 1,000, 10,000, 100,000, or 500,000 IU/mL. Probit analysis resulted in a LD10 (lethal dose for 10% of injected fish) of 52,868 IU/mL (95% confidence limits: 31,522 - 69,490 IU/mL) and a LD50 of 131,466 IU/mL (113,095 - 157,878 IU/mL). Brood stock were injected (i.p.) with 200 μL of penicillin at concentrations of 0 (control), 10,000, 50,000, 100,000, 200,000, 400,000, or 800,000 IU/mL. Mortality ranged from 0% to 7% (50,000 IU/mL treatment) and did not significantly differ among treatments. In 28 h exposure tests, penicillin concentrations of ≥ 333 IU/mL were required to completely suppress growth of F. psychrophilum. In 15 min exposures, ≥ 10,000 IU/mL were needed to achieve the same result. Tests indicated that rainbow trout eggs can tolerate 1 h exposures to penicillin-G concentrations as high as 100,000 IU/mL, and that brood fish can tolerate injections of at least 800,000 IU/mL. Fingerling data however, suggested that injection of doses greater than 10,000 IU/mL can be toxic. The data suggests rainbow trout eggs or brood can tolerate high doses of penicillin-G that could be used for controlling the transfer of F. psychrophilum to hatcheries receiving eggs.

The use of warmed water treatment to induce protective immunity against the bacterial cold-water disease pathogen Flavobacterium psychrophilum in ayu ( Plecoglossus altivelis)

We investigated the induction of protective immunity against bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum by warmed water treatment in ayu ( Plecoglossus altivelis). Fish were immersed in a live bacterial suspension (10 7 CFU mL −1) for 30 min and placed in 700 L concrete tanks. The 28 °C warmed water treatment lasted 3 days and began 1, 6, and 24 h after immersion in the live bacterial suspension. A naïve control fish group was immersed in a sterilized modified Cytophaga (MCY) broth instead of the bacterial suspension. Fourteen days after the immersion, agglutination antibody titers against F. psychrophilum were measured by using micro-titer methods. Fish were then exposed to a bacterial bath to infect them with live F. psychrophilum, and cumulative mortality was monitored. Fish treated with warmed water at 1, 6, and 24 h after immersion in the live bacterial suspension had cumulative mortalities of 36%, 30%, and 18%, respectively, all of which were significantly lower than the cumulative mortality of the naïve control fish (90%). Treated fish also showed high antibody titers against F. psychrophilum in agglutination tests. These results demonstrate that warmed water treatment could not only cure BCWD but also immunize the fish against the causative agent F. psychrophilum.

Identification of immunogenic proteins within distinct molecular mass fractions of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the aetiological agent of bacterial coldwater disease (CWD), and this pathogen has large economic impacts on salmonid aquaculture worldwide. Previously, it was demonstrated that high levels of protection against F. psychrophilum challenge were conferred to rainbow trout, Oncorhynchus mykiss (Walbaum), by immunization with distinct molecular mass fractions of the bacterium, and specific antibodies were correlated with protection. In this study, an immunoproteomic analysis of F. psychrophilum was performed using two-dimensional polyacrylamide gel electrophoresis and Western blotting with serum from fish immunized with high- and mid-molecular mass fractions of the bacterium. Mass spectrometry was used to determine the protein identity, and 15 immunogenic proteins were positively identified following Mascot searches of the F. psychrophilum genome. Based on known function and immunogenicity of homologous proteins in other bacterial pathogens, antibodies specific for several of the identified proteins may be important for protective immunity from CWD. These include outer membrane protein OmpA (P60), trigger factor, ClpB, elongation factor G, gliding motility protein GldN and a conserved hypothetical protein. This work increases the understanding of the protective humoral immune response of rainbow trout against these distinct molecular mass fractions of F. psychrophilum and provides new potential targets for recombinant protein vaccine development.

Quantification of Flavobacterium psychrophilum in rainbow trout, Oncorhynchus mykiss (Walbaum), tissues by qPCR

A qPCR assay was developed for rapid and sensitive detection of Flavobacterium psychrophilum, the aetiological agent of bacterial cold-water disease and rainbow trout fry syndrome in salmonid fish worldwide. A set of F. psychrophilum-specific primers based on 16S rRNA gene sequences was designed and validated for specific detection and quantification of DNA isolated from representative strains of F. psychrophilum. The qPCR assay exhibited a high specificity for the 16S rRNA gene of F. psychrophilum (from 4 × 10(8) down to 11 copies per reaction) but not for other Flavobacterium species or other bacteria including fish pathogens. This qPCR-based method proved to be useful in the quantification of the F. psychrophilum titre present within organs dissected out from diseased fish. As the F. psychrophilum genome contains six copies of the 16S rRNA gene, we could infer a limit of detection corresponding to two bacteria per reaction, corresponding to 800 bacteria per fish tissue sample, and therefore 20 F. psychrophilum cells mg(-1) of tissue (for sample weighing 40 mg). The qPCR assay reported here could be a useful tool for veterinary diagnostic laboratories to monitor the F. psychrophilum infection level in fish farms.

Enhanced resistance to coldwater disease following feeding of probiotic bacterial strains to rainbow trout ( Oncorhynchus mykiss)

Flavobacterium psychrophilum is the causative agent of coldwater disease (CWD), also called rainbow trout fry syndrome (RTFS), and has a major impact on salmonid aquaculture worldwide. The aim of this study was to evaluate the efficacy of non-pathogenic probiotic bacterial strains to reduce mortality due to F. psychrophilum infection in rainbow trout, ( Oncorhynchus mykiss). Candidate probiotics, previously demonstrated to have in vitro inhibition towards F. psychrophilum and the potential to survive in the gastrointestinal (GI) tract, were administered through oil-dressed feed at 10 6 to 10 8 bacterial cells g − 1 , prior to and/or after infection with F. psychrophilum. Administration of two of the ten probiotic strains (C6-6 and C6-8), resulted in a significant reduction in mortality due to F. psychrophilum infection ( P < 0.05) when compared to control fish. Both probiotic strains were consistently reisolated from the GI tract indicating their ability to persistently colonize the GI tract. The 16S rRNA sequences from strains C6-6 and C6-8 indicated that these strains were distinct from each other but shared 99.8% similarity with Enterobacter amnigenus and Enterobacter strain PIC15, respectively. Results from this study demonstrate the potential for these probiotic strains to provide benefit and an alternative strategy for managing F. psychrophilum infections in fish in aquaculture.

A Review of Flavobacterium Psychrophilum Biology, Clinical Signs, and Bacterial Cold Water Disease Prevention and Treat

Bacterial coldwater disease and other infections caused by Flavobacterium psychrophilum are a worldwide concern, particularly for freshwater salmonid hatcheries. F. psychrophilum infections can be difficult to control; antibiotic resistance is common and no effective vaccines are currently available. This review summarizes the biology and charac- teristics of this important pathogen, as well as the techniques required for isolation and identification. In addition, the epi- demiology, clinical signs, treatment, and possible preventative measures of bacterial coldwater disease are discussed.

Identification of Cold-Temperature-Regulated Genes in Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS). It causes disease primarily in fresh water-reared salmonids, but other fish species can also be affected. A diverse array of clinical conditions is associated with BCWD, including tail rot (peduncle disease), necrotic myositis, and cephalic osteochondritis. Degradation of connective and muscular tissues by extracellular proteases is common to all of these presentations. There are no effective vaccines to prevent BCWD or RTFS, and antibiotics are often used to prevent and control disease. To identify virulence factors that might permit development of an efficacious vaccine, cDNA suppression subtractive hybridization (SSH) was used to identify cold-regulated genes in a virulent strain of F. psychrophilum. Genes predicted to encode a two-component system sensor histidine kinase (LytS), an ATP-dependent RNA helicase, a multidrug ABC transporter permease/ATPase, an outer membrane protein/protective antigen OMA87, an M43 cytophagalysin zinc-dependent metalloprotease, a hypothetical protein, and four housekeeping genes were upregulated at 8°C versus the level of expression at 20°C. Because no F. psychrophilum gene was known to be suitable as an internal standard in reverse transcription-quantitative real-time PCR (RT-qPCR) experiments, the expression stability of nine commonly used reference genes was evaluated at 8°C and 20°C. Expression of the 16S rRNA was equivalent at both temperatures, and this gene was used in RT-qPCR experiments to verify the SSH findings. With the exception of the ATCC 49513 strain, similar patterns of gene expression were obtained with 11 other representative strains of F. psychrophilum.

The Prevalence of Flavobacterium psychrophilum among Wild Ayu in Rivers that Do Not Have a History of Ayu Stocking

We examined the prevalence of Flavobacterium psychrophilum infection in wild ayu Plecoglossus altivelis in four rivers in Miyagi Prefecture that have not been artificially stocked with juvenile ayu. The isolation rates of F. psychrophilum from apparently healthy ayu ranged from 58 to 100% (mean 89%). The majority of isolates were classified into the A/S genotype. All of the examined isolates were highly virulentto juvenile ayu. Our results suggested that bacterial cold water disease was present in wild ayu populations, even in rivers without a history of ayu stocking.

Low Likelihood of Intra-Ovum Infection with Flavobacterium psychrophilum in Cultured Salmonids in Japan

We evaluated the occurrence of intra-ovum infection with Flavobacterium psychrophilum, the causative agent of bacterial cold-water disease (BCWD) and rainbow trout fry syndrome (RTFS), in the eggs of six salmonid species in Japan. Flavobacterium psychrophilum was isolated from four eggs in one of 13 lots of eyed eggs (999 eggs) imported from the United States. In contrast, the bacterium was not isolated from 28 lots (1,680 eggs) or 61 lots (3,693 eggs) of domestically produced unfertilized or eyed eggs, respectively. Our results indicate there is a low likelihood of intra-ovum infection in cultured salmonids in Japan.

Disinfection of Contaminated Equipment: Evaluation of Benzalkonium Chloride Exposure Time and Solution Age and the Ability of Air‐Drying to Eliminate Flavobacterium psychrophilum

Disinfection of equipment that comes in contact with fish can help to minimize the spread of Flavobacterium psychrophilum (the etiological agent of bacterial coldwater disease) within and among fish culture facilities. We present the results of three studies that evaluated the potential use of benzalkonium chloride and air-drying to kill surface-attached F. psychrophilum. In the first study, we established a vat with a 600-mg/L benzalkonium chloride solution and sampled this solution 0, 14, 35, 56, 70, and 84 d after creation. The solution was kept outdoors and subjected to typical hatchery use. Plastic test strips were dipped in a solution containing F. psychrophilum and were then immersed in benzalkonium chloride for 0, 1, 10, 30, or 60 min. The strips were then rinsed with sterile water and streaked across a plate containing tryptone yeast extract salts (TYES) medium. No culturable bacteria were detected from any strips immersed for 10, 30, or 60 min. Bacteria were detected on 17% of the strips that were immersed for 1 min. The age of the benzalkonium chloride solution had no effect on disinfection ability. In the second study, plastic strips were immersed in a solution containing F. psychrophilum and then were dipped in a 600-mg/L benzalkonium chloride solution for 10 s. The strips were then air-dried for 1 h and were streaked onto TYES medium. No bacterial growth was observed from any strips in the second experiment. The third study determined whether air-drying alone was sufficient to kill F. psychrophilum. Plastic strips were dipped in a solution containing F. psychrophilum; were allowed to dry at room temperature for 0, 24, 48, or 96 h; and were then streaked across TYES medium. Bacteria were cultured from strips representing each drying interval, indicating that air-drying times of 96 h or less are insufficient to kill F. psychrophilum.

Rapid and highly sensitive detection of Flavobacterium psychrophilum using high gradient immunomagnetic separation with flow cytometry

The bacterial cold water disease caused by Flavobacterium psychrophilum is a concern not only for the aquaculture industry, but also the environment. We developed a novel method for detecting F. psychrophilum using high gradient magnetic separation (HGMS) with flow cytometry (FCM). HGMS is a magnetic separation method in which the magnetic force is strengthened by introducing a magnetic gradient between the magnetic filter and nearby column. F. psychrophilum cells that had reacted with immunomagnetic beads were loaded onto the column containing the magnetic filters. When the column was placed inside a neodymium ring magnet, the cells became stuck to the magnetic filters. When magnetic separation of a 500 ml sample was performed, more than 80% of the beads were trapped by the magnetic filter. The collected cells were easily removed from the filter by turning off the magnetic field. The cells were reacted with fluorescein isothiocyanate-labeled antibody, and then fluorescence was measured using FCM. The detection sensitivity was three orders of magnitude higher than that of an FCM assay without HGMS. The cell numbers determined by FCM correlated with those obtained using the colony counting method in the range of approximately 10 1–10 5 colony forming units per milliliter. One FCM assay could be completed within 60 s and the total assay time, including sample preparation, was less than 2.5 h. The proposed method using HGMS and FCM allows for rapid and highly sensitive detection of F. psychrophilum.

Evidence of major genes affecting resistance to bacterial cold water disease in rainbow trout using Bayesian methods of segregation analysis1

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. We previously detected genetic variation for BCWD resistance in our rainbow trout population, and a family-based selection program to improve resistance was initiated at the National Center for Cool and Cold Water Aquaculture (NCCCWA). This study investigated evidence of major trait loci affecting BCWD resistance using only phenotypic data (without using genetic markers) and Bayesian methods of segregation analysis (BMSA). A total of 10,603 juvenile fish from 101 full-sib families corresponding to 3 generations (2005, 2007, and 2009 hatch years) of the NCCCWA population were challenged by intraperitoneal injection with Flavobacterium psychrophilum, the bacterium that causes BCWD. The results from single- and multiple-QTL models of BMSA suggest that 6 to 10 QTL explaining 83 to 89% of phenotypic variance with either codominant or dominant disease-resistant alleles plus polygenic effects may underlie the genetic architecture of BCWD resistance. This study also highlights the importance of polygenic background effects in the genetic variation of BCWD resistance. The polygenic heritability on the observed scale of survival status is slightly larger than that previously reported for rainbow trout BCWD resistance. These findings provide the basis for designing informative crosses for QTL mapping and carrying out genome scans for QTL affecting BCWD resistance in rainbow trout.

The bacterial community associated with the leechMyzobdella lugubrisLeidy 1851 (Hirudinea: Piscicolidae) from Lake Erie, Michigan, USA

Leeches are widespread in the Great Lakes Basin, yet their potential to harbor disease-causing agents has not been investigated. The purpose of this study was to identify the bacterial community of the commonly occurring leech, Myzobdella lugubris, within the Lake Erie Watershed. Leech samples were collected from the pectoral fins of channel catfish, Ictalurus punctatus, and freshwater drum, Aplodinotus grunniens, from Lake Erie in commercial trap nets and pooled into two samples based on host attachment. Bacteria from within the viscera of M. lugubris were identified by sequencing their 16S rRNA (rDNA) gene of amplified community bacterial DNA extracted from pooled leech homogenate samples and were checked for similarity in two public databases: the Ribosomal Database Project and BLAST. Bacteria belonging to the phylum Bacteroidetes, beta-proteobacteria, Verrucomicrobia, and unclassified Bacteria were present in the leech samples. A large number of bacteria found within leeches attached to channel catfish consisted of sequences that could not be classified beyond the Domain Bacteria. However, many of these sequences were homologous (< 45%) to the phylum Bacteroidetes. One of the five genera detected in the leech homogenates was Flavobacterium psychrophilum, a serious fish pathogen that causes Bacterial Cold Water Disease. While the occurrence of genera varies, bacteria associated with the two fish species were similar.

Relationships between growth and disease resistance in rainbow trout, Oncorhynchus mykiss (Walbaum)

Rainbow trout from 23 families were evaluated for growth and resistance to the bacterial coldwater disease (BCWD) caused by Flavobacterium psychrophilum and infectious haematopoietic necrosis (IHN) caused by IHN virus. Average family weights were between 161 and 263 g with an average of 225 g at 213 days post-fertilization with specific growth rates ranging from 2.37 to 2.88. Per cent survival of fish challenged with F. psychrophilum was between 18% and 100%, while for those challenged with IHNV, the range was between 12% and 93%. Significant positive correlations were found for end body weight and resistance to IHN (P < 0.05) and for early body weight and resistance to BCWD (P < 0.1). However, no significant correlations were detected between resistance to both pathogens or disease resistance and overall genetic diversity or diversity within the major histocompatibility locus.

Genetic diversity and antimicrobial resistance of Flavobacterium psychrophilum isolated from cultured rainbow trout, Onchorynchus mykiss (Walbaum), in Spain

Flavobacterium psychrophilum is the causative agent of bacterial cold-water disease (CWD) and rainbow trout fry syndrome (RTFS) in salmonids. These diseases are a major problem in the aquaculture industry in Spain, and a better understanding of the epidemiology of F. psychrophilum isolates is necessary to improve management strategies. In this study, to investigate genetic variability of this bacterium, pulsed-field gel electrophoresis after DNA digestion with endonuclease StuI, plasmid profiling analysis and antimicrobial susceptibility testing were undertaken with 25 isolates of F. psychrophilum from Spain. These isolates were classified into 17 patterns by PFGE analysis, which were grouped into four clusters and seven independent branches. Twenty isolates (80%) possessed plasmids of 3.5 kb (n = 13) or 5.5 kb (n = 7). No plasmids were associated with antibiotic resistance to oxytetracycline (OTC) or florfenicol (FLO). Twenty isolates (80%) had minimum inhibitory concentrations (MICs) to OTC of between 2.4 and 9.7 microg mL(-1), and all isolates were susceptible to FLO. A relationship between the origin of the isolates and PFGE genotypes was found. Plasmid profile typing correlated with PFGE profile typing, whereas no correlation was found between antimicrobial susceptibility testing and PFGE profiles. These results suggest that the population of F. psychrophilum with pathogenic potential in northern Spain is quite heterogeneous.

Antimicrobial Susceptibility of Flavobacterium psychrophilum Isolates from Ontario

Flavobacterium psychrophilum is the etiological agent of bacterial coldwater disease (BCWD), an important disease in the Ontario fish farming industry and in finfish aquaculture in temperate waters worldwide. The development of antimicrobial resistance by F. psychrophilum is a concern because management of outbreaks of BCWD often requires the use of antibiotics. Seventy-two isolates of F. psychrophilum collected over a 16-year period from farmed salmonids with clinical signs of BCWD were tested for susceptibility to 10 antimicrobial agents using cation-adjusted Mueller-Hinton broth in custom Trek Sensititre susceptibility plates for aquaculture. The minimum inhibitory concentrations (MICs) for the isolates were determined by means of a broth microdilution antimicrobial susceptibility testing method established by the Clinical and Laboratory Standards Institute. Most of the F. psychrophilum isolates had decreased susceptibility to two of the four antibiotics licensed for use in Ontario (i.e., ormetoprim-sulfadimethoxine [> or =0.5/9.5 .tg/mL for 93% of isolates] and trimethoprim-sulfamethoxazole [> or = 0.25/4.8 microg/mL for 89% of isolates]). High MIC values (> or =2 microg/mL) were obtained for florfenicol and oxytetracycline in 53% and 61% of the isolates, respectively, and 83% of the isolates were relatively susceptible (< or =16 microg/mL) to erythromycin. The MIC values were also high for ampicillin, oxolinic acid, and gentamicin.

Response to selection for bacterial cold water disease resistance in rainbow trout1,2

A family-based selection program was initiated at the National Center for Cool and Cold Water Aquaculture in 2005 to improve resistance to bacterial cold water disease (BCWD) in rainbow trout. The objective of this study was to estimate response to 2 generations of selection. A total of 14,841 juvenile fish (BW = 3.1 g; SD = 1.1 g) from 230 full-sib families and 3 randomly mated control lines were challenged intraperitoneally with Flavobacterium psychrophilum, the bacterium that causes BCWD, and mortalities were observed for 21 d. Selection was applied to family EBV derived from a proportional-hazards frailty (animal) model while constraining rate of inbreeding to <or=1% per generation. After adjusting for nongenetic effects, survival rate of select-line families increased by 24.6 +/- 6.8 and 44.7 +/- 6.7 (cumulative) percentage points after 1 and 2 generations of selection, respectively (P < 0.01). Genetic trend, estimated from a linear animal model that fit genetic group effects, was 19.0 +/- 4.1 percentage points per generation and approached significance (P = 0.07). Heritability estimates from the proportional-hazards frailty model and linear animal model were similar (0.22 and 0.23, respectively), and family EBV from both models were highly correlated (-0.92). Accuracy of selection, estimated as the correlation between mid-parent EBV and progeny survival rate, was 0.20 (P < 0.01) for the proportional-hazards frailty model and 0.18 (P = 0.01) for the linear animal model. Accuracy estimates were not different (P = 0.81) between the models. This study demonstrates that selective breeding can be effective for improving resistance to experimental BCWD challenge in rainbow trout.

Viable but Non-culturable State of Bacterial Cold-water Disease Pathogen Flavobacterium psychrophilum at Various Temperatures

Bacterial cold-water disease (BCWD) caused by Flavobacterium psychrophilum leads to heavy mortality of ayu Plecoglossus altivelis in Japan. Previously, a 28°C warmed water treatment was shown to be an effective treatment for BCWD, since F. psychrophilum can not grow and maintain colonies at this temperature. However, it was unclear whether the bacteria might resuscitate after the treatment was over. Therefore, we investigated colony formation, membrane potential and pathogenicity of F. psychrophilum at various temperatures (15°C, 23°C, 28°C and 33°C) in sterilized underground water. Within 2 days at 28°C, F. psychrophilum completely lost their colony-forming abilities but still maintained their membrane potentials. It seemed that these cells entered into viable but non-culturable (VBNC) state. However, experimental ayu infection revealed that VBNC F. psychrophilum cells were unable to cause BCWD, suggesting that the cells were progressing towards death at 28°C.

Development and evaluation of a multiplex PCR assay for simultaneous detection ofFlavobacterium psychrophilum,Yersinia ruckeriandAeromonas salmonicidasubsp.salmonicidain 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.

Mode of the Intra-ovum Infection of Flavobacterium psychrophilum in Salmonid Eggs

The mode of intra-ovum infection by Flavobacterium psychrophilum, which causes bacterial cold-water disease and rainbow trout fry syndrome, was investigated in eggs of rainbow trout Oncorhynchus mykiss. When newly spawned unfertilized eggs were immersion-challenged with F. psychrophilum at concentrations of 107 CFU/mL or higher and subsequently inseminated and water-hardened, the bacteria were detected in the egg contents. The infection rate in eggs was related to the bacterial concentration in a dose-dependent manner. When mature female rainbow trout were intraperitoneally injected with F. psychrophilum (3.3 × 109 CFU/fish) 5-9 days prior to ovulation, intra-ovum infection was found in eggs spawned from the fish showing heavy contamination of the bacterium in the ovarian fluid (> 106 CFU/mL). These results indicate that F. psychrophilum can contaminate the surface of eggs in ovarian fluid and enter eggs during water-hardening. Live bacterial counts within experimentally infected eggs and immuno-staining observations on the eggs suggested that F. psychrophilum passively entered into the eggs (< 10 CFU/egg) and subsequently grew to over 107 CFU/egg in the perivitelline space by the eyed egg stage. Multiplication of the bacterium in the perivitelline space did not affect the survival of eggs.