Varying Flavobacterium psychrophilum shedding dynamics in three bacterial coldwater disease-susceptible salmonid (Family Salmonidae) species.

Flavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and is responsible for substantial losses in farm and hatchery-reared salmonids (Family Salmonidae). Although F. psychrophilum infects multiple economically important salmonids and is transmitted horizontally, the extent of knowledge regarding F. psychrophilum shedding rates and duration is limited to rainbow trout (Oncorhynchus mykiss). Concurrently, hundreds of F. psychrophilum sequence types (STs) have been described using multilocus sequence typing (MLST), and evidence suggests that some variants have distinct phenotypes, including differences in host associations. Whether shedding dynamics differ among F. psychrophilum variants and/or salmonids remains unknown. Thus, three F. psychrophilum isolates (e.g., US19, US62, and US87) in three MLST STs (e.g., ST13, ST277, and ST275) with apparent host associations for coho salmon (O. kisutch), Atlantic salmon (Salmo salar), or rainbow trout were intramuscularly injected into each respective fish species. Shedding rates of live and dead fish were determined by quantifying F. psychrophilum loads in water via quantitative PCR. Both live and dead Atlantic and coho salmon shed F. psychrophilum, as did live and dead rainbow trout. Regardless of salmonid species, dead fish shed F. psychrophilum at higher rates (e.g., up to ~108-1010 cells/fish/hour) compared to live fish (up to ~107-109 cells/fish/hour) and for a longer duration (5-35 days vs 98 days); however, shedding dynamics varied by F. psychrophilum variant and/or host species, a matter that may complicate BCWD management. Findings herein expand knowledge on F. psychrophilum shedding dynamics across multiple salmonid species and can be used to inform future BCWD management strategies.IMPORTANCEFlavobacterium psychrophilum causes bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, both of which cause substantial losses in farmed and hatchery-reared salmon and trout populations worldwide. This study provides insight into F. psychrophilum shedding dynamics in rainbow trout (Oncorhynchus mykiss) and, for the first time, coho salmon (O. kisutch) and Atlantic salmon (Salmo salar). Findings revealed that live and dead fish of all fish species shed the bacterium. However, dead fish shed F. psychrophilum at higher rates than living fish, emphasizing the importance of removing dead fish in farms and hatcheries. Furthermore, shedding dynamics may differ according to F. psychrophilum genetic variant and/or fish species, a matter that may complicate BCWD management. Overall, study results provide deeper insight into F. psychrophilum shedding dynamics and will guide future BCWD management strategies.

Phenotypic and Genetic Characterization of Flavobacterium psychrophilum Recovered from Diseased Salmonids in China.

Flavobacterium psychrophilum, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome, causes great economic losses in salmonid aquaculture worldwide. Recent molecular studies have uncovered important epidemiological and ecological aspects of this pathogen; however, such data are lacking for F. psychrophilum populations affecting aquaculture in China. Herein, F. psychrophilum phenotype, genotype, and virulence were characterized for isolates recovered from epizootics in multiple salmonid aquaculture facilities across China. Thirty-one F. psychrophilum isolates, originating from four provinces and three host fish species, were predominantly homogeneous biochemically but represented 5 sequence types (STs) according to multilocus sequence typing (MLST) that belonged to clonal complex CC-ST10 or 3 newly recognized singleton STs. PCR-based serotyping classified 19 and 12 F. psychrophilum isolates into molecular serotypes 1 and 0, respectively, showing an obvious relationship with host species. Antimicrobial susceptibility analysis via broth microdilution revealed reduced susceptibility to enrofloxacin, flumequine, and oxolinic acid, moderate susceptibility to gentamicin, erythromycin, and florfenicol, and variable susceptibility to ampicillin and oxytetracycline. In vivo challenge experiments confirmed the ability of two representative Chinese F. psychrophilum isolates to induce typical signs of BCWD and mortality in 1-year-old rainbow trout (Oncorhynchus mykiss). Findings collectively demonstrate (i) that BCWD outbreaks in China studied thus far are caused by F. psychrophilum lineages that are common on other continents (e.g., CC-ST10) and others that have not been reported elsewhere (e.g., ST355, ST356, ST357), (ii) that F. psychrophilum molecular serotypes distinguish isolates from different host fish species, even within STs, and (iii) reduced F. psychrophilum antimicrobial susceptibility against compounds used for BCWD control in China. IMPORTANCE Flavobacterium psychrophilum causes substantial economic losses in salmonid aquaculture worldwide. Although this bacterium is also believed to be a disease source in China, published reports of its presence do not yet exist. Herein, F. psychrophilum was linked to multiple disease outbreaks in several salmonid aquaculture facilities within four Chinese provinces, and polyphasic characterization revealed that most isolates were genetically distinct from strains recovered on other continents. Analyses further revealed the predominating molecular serotypes, antimicrobial susceptibility profiles, and pathogenic potential of two representative recovered isolates. Collectively, the results presented here provide important data on the epidemiology and disease ecology of F. psychrophilum in China and pave the way for targeted prevention and control methods to be pursued in the future.

Detection of selection signatures in farmed coho salmon (Oncorhynchus kisutch) using dense genome-wide information.

Animal domestication and artificial selection give rise to gradual changes at the genomic level in populations. Subsequent footprints of selection, known as selection signatures or selective sweeps, have been traced in the genomes of many animal livestock species by exploiting variation in linkage disequilibrium patterns and/or reduction of genetic diversity. Domestication of most aquatic species is recent in comparison with land animals, and salmonids are one of the most important fish species in aquaculture. Coho salmon (Oncorhynchus kisutch), cultivated primarily in Chile, has been subjected to breeding programs to improve growth, disease resistance traits, and flesh color. This study aimed to identify selection signatures that may be involved in adaptation to culture conditions and traits of productive interest. To do so, individuals of two domestic populations cultured in Chile were genotyped with 200 thousand SNPs, and analyses were conducted using iHS, XP-EHH and CLR. Several signatures of selection on different chromosomal regions were detected across both populations. Some of the identified regions under selection contained genes such anapc2, alad, chp2 and myn, which have been previously associated with body weight in Atlantic salmon, or sec24d and robo1, which have been associated with resistance to Piscirickettsia salmonis in coho salmon. Findings in our study can contribute to an integrated genome-wide map of selection signatures, to help identify the genetic mechanisms of phenotypic diversity in coho salmon.

Comparison of infectious agents detected from hatchery and wild juvenile Coho salmon in British Columbia, 2008-2018.

Infectious diseases are potential contributors to decline in Coho salmon (Oncorhynchus kisutch) populations. Although pathogens are theoretically considered to pose higher risk in high-density rearing environments like hatcheries, there is no direct evidence that hatchery-origin Coho salmon increase the transmission of infectious agents to sympatric wild populations. This study was undertaken to compare prevalence, burden, and diversity of infectious agents between hatchery-reared and wild juvenile Coho salmon in British Columbia (BC), Canada. In total, 2,655 juvenile Coho salmon were collected between 2008 and 2018 from four regions of freshwater and saltwater in BC. High-throughput microfluidics qPCR was employed for simultaneous detection of 36 infectious agents from mixed-tissue samples (gill, brain, heart, liver, and kidney). Thirty-one agents were detected at least once, including ten with prevalence >5%. Candidatus Brachiomonas cysticola, Paraneuclospora theridion, and Parvicapsula pseudobranchiocola were the most prevalent agents. Diversity and burden of infectious agents were substantially higher in marine environment than in freshwater. In Mainland BC, infectious burden and diversity were significantly lower in hatchery smolts than in wild counterparts, whereas in other regions, there were no significant differences. Observed differences in freshwater were predominantly driven by three parasites, Loma salmonae, Myxobolus arcticus, and Parvicapsula kabatai. In saltwater, there were no consistent differences in agent prevalence between hatchery and wild fish shared among the west and east coasts of Vancouver Island. Although some agents showed differential infectious patterns between regions, annual variations likely contributed to this signal. Our findings do not support the hypothesis that hatchery smolts carry higher burdens of infectious agents than conspecific wild fish, reducing the potential risk of transfer to wild smolts at this life stage. Moreover, we provide a baseline of infectious agents in juvenile Coho salmon that will be used in future research and modeling potential correlations between infectious profiles and marine survival.

Genomic Predictions and Genome-Wide Association Study of Resistance Against Piscirickettsia salmonis in Coho Salmon (Oncorhynchus kisutch) Using ddRAD Sequencing.

Piscirickettsia salmonis is one of the main infectious diseases affecting coho salmon (Oncorhynchus kisutch) farming, and current treatments have been ineffective for the control of this disease. Genetic improvement for P. salmonis resistance has been proposed as a feasible alternative for the control of this infectious disease in farmed fish. Genotyping by sequencing (GBS) strategies allow genotyping of hundreds of individuals with thousands of single nucleotide polymorphisms (SNPs), which can be used to perform genome wide association studies (GWAS) and predict genetic values using genome-wide information. We used double-digest restriction-site associated DNA (ddRAD) sequencing to dissect the genetic architecture of resistance against P. salmonis in a farmed coho salmon population and to identify molecular markers associated with the trait. We also evaluated genomic selection (GS) models in order to determine the potential to accelerate the genetic improvement of this trait by means of using genome-wide molecular information. A total of 764 individuals from 33 full-sib families (17 highly resistant and 16 highly susceptible) were experimentally challenged against P. salmonis and their genotypes were assayed using ddRAD sequencing. A total of 9,389 SNPs markers were identified in the population. These markers were used to test genomic selection models and compare different GWAS methodologies for resistance measured as day of death (DD) and binary survival (BIN). Genomic selection models showed higher accuracies than the traditional pedigree-based best linear unbiased prediction (PBLUP) method, for both DD and BIN. The models showed an improvement of up to 95% and 155% respectively over PBLUP. One SNP related with B-cell development was identified as a potential functional candidate associated with resistance to P. salmonis defined as DD.

Genetic Diversity of Flavobacterium psychrophilum Isolates from Three Oncorhynchus spp. in the United States, as Revealed by Multilocus Sequence Typing.

UNLABELLED: The use of a multilocus sequence typing (MLST) technique has identified the intraspecific genetic diversity of U.S. Flavobacterium psychrophilum, an important pathogen of salmonids worldwide. Prior to this analysis, little U.S. F. psychrophilum genetic information was known; this is of importance when considering targeted control strategies, including vaccine development. Herein, MLST was used to investigate the genetic diversity of 96 F. psychrophilum isolates recovered from rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), and Chinook salmon (Oncorhynchus tshawytscha) that originated from nine U.S. states. The isolates fell into 34 distinct sequence types (STs) that clustered in 5 clonal complexes (CCs) (n = 63) or were singletons (n = 33). The distribution of STs varied spatially, by host species, and in association with mortality events. Several STs (i.e., ST9, ST10, ST30, and ST78) were found in multiple states, whereas the remaining STs were localized to single states. With the exception of ST256, which was recovered from rainbow trout and Chinook salmon, all STs were found to infect a single host species. Isolates that were collected during bacterial cold water disease outbreaks most frequently belonged to CC-ST10 (e.g., ST10 and ST78). Collectively, the results of this study clearly demonstrate the genetic diversity of F. psychrophilum within the United States and identify STs of clinical significance. Although the majority of STs described herein were novel, some (e.g., ST9, ST10, ST13, ST30, and ST31) were previously recovered on other continents, which demonstrates the transcontinental distribution of F. psychrophilum genotypes. IMPORTANCE: Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is an important bacterial pathogen of wild and farmed salmonids worldwide. These infections are responsible for large economic losses globally, yet the genetic diversity of this pathogen remains to be fully investigated. Previous studies have identified the genetic diversity of this pathogen in other main aquaculture regions; however, little effort has been focused on the United States. In this context, this study aims to examine the genetic diversity of F. psychrophilum from the United States, as this region remains important in salmonid aquaculture.

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

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

Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout, Oncorhynchus mykiss (Walbaum), and spawning coho salmon, O. kisutch (Walbaum).

Flavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. This study examined the genetic diversity of F. psychrophilum isolates retrieved from multiple epizootics at rainbow trout, Oncorhynchus mykiss, rearing facilities and from spawning coho salmon, O. kisutch. A total of 139 isolates were confirmed as F. psychrophilum by PCR assay and were further typed using pulsed-field gel electrophoresis (PFGE). Multiple epizootics at three proximally located rainbow trout rearing facilities were numerically dominated by three PFGE profiles, which accounted for 76% of all trout isolates. In coho salmon, 19 PFGE profiles were differentiated by PFGE and four numerically dominant PFGE profiles represented 56% of all coho salmon isolates. PFGE analysis also indicated that the average similarity of macrorestriction patterns of F. psychrophilum isolates was greater in rainbow trout than in coho salmon (88% vs. 70%). Furthermore, it was not unusual to isolate multiple PFGE profiles from a single coho salmon sample whereas only two PFGE profiles were shared between two sample dates separated by 1 month. It is clear that the domestic rainbow trout aquaculture facilities studied here were primarily affected by a complex of genetically related strains whereas spawning coho salmon supported a much more genetically diverse collection of F. psychrophilum.

16S rDNA-based analysis of dominant bacterial populations associated with early life stages of coho salmon (Oncorhynchus kisutch).

In this study, we used a 16S rDNA-based approach to determine bacterial populations associated with coho salmon (Oncorhynchus kisutch) in its early life stages, highlighting dominant bacteria in the gastrointestinal tract during growth in freshwater. The present article is the first molecular analysis of bacterial communities of coho salmon. Cultivability of the salmon gastrointestinal microbiota was estimated by comparison of direct microscopic counts (using acridine orange) with colony counts (in tryptone soy agar). In general, a low fraction (about 1%) of the microbiota could be recovered as cultivable bacteria. Using DNA extracted directly from individuals belonging to the same lot, bacterial communities present in eggs and gastrointestinal tract of first-feeding fries and juveniles were monitored by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). The DGGE profiles revealed simple communities in all stages and exposed changes in bacterial community during growth. Sequencing and phylogenetic analysis of excised DGGE bands revealed the nature of the main bacteria found in each stage. In eggs, the dominant bacteria belonged to beta-Proteobacteria (Janthinobacterium and Rhodoferax). During the first feeding stage, the most abundant bacteria in the gastrointestinal tract clustered with gamma-Proteobacteria (Shewanella and Aeromonas). In juveniles ranging from 2 to 15 g, prevailing bacteria were Pseudomonas and Aeromonas. To determine the putative origin of dominant Pseudomonas and Aeromonas found in juvenile gastrointestinal tracts, primers for these groups were designed based on sequences retrieved from DGGE gel. Subsequently, samples of the water influent, pelletized feed, and eggs were analyzed by PCR amplification. Only those amplicons obtained from samples of eggs and the water influent presented identical sequences to the dominant bands of DGGE. Overall, our results suggest that a stable microbiota is established after the first feeding stages and its major components could be derived from water and egg epibiota.

Multiplex PCR for simultaneous detection of five virulence hemolysin genes in Vibrio anguillarum.

A multiplex PCR was developed for detection of hemolysin-producing Vibrio anguillarum using primers targeting five hemolysin genes (vah1, vah2, vah3, vah4 and vah5). This method was successful in amplifying reactions containing as little as 100 fg of genomic template DNA. The direct detection of V. anguillarum in clinical specimens by this multiplex PCR was also successful in reactions containing as few as 10 bacterial cells. This multiplex PCR method can be a rapid and sensitive method for detecting pathogenic V. anguillarum.

Isolation and identification of Photobacterium phosphoreum from an unexpected niche: migrating salmon.

Six luminous bacteria were isolated from migrating salmon in the Yukon River, Alaska. All isolates were identified as Photobacterium phosphoreum. Previous studies suggest that P. phosphoreum is an exclusively marine bacterium, while our Alaskan isolates are from salmon which migrated up to 1,228 km from the marine environment.

Immunoresponse of Coho salmon immunized with a gene expression library from Piscirickettsia salmonis.

We have used the expression library immunization technology to study the protection of Coho salmon Oncorhynchus kisutch to the infection with Piscirickettsia salmonis. Purified DNA from this bacterium was sonicated and the fragments were cloned in the expression vector pCMV-Bios. Two libraries were obtained containing 22,000 and 28,000 colonies and corresponding to approximately 8 and 10 times the genome of the pathogen, respectively. On average, the size of the inserts ranged between 300 and 1,000 bp. The plasmid DNA isolated from one of these libraries was purified and 20 micrograms were injected intramuscularly into 60 fish followed by a second dose of 10 micrograms applied 40 days later. As control, fish were injected with the same amount of DNA of the vector pCMV-Bios without insert. The titer of IgM anti-P. salmonis of vaccinated fish, evaluated 60 days post-injection, was significantly higher than that of the control group injected with the vector alone. Moreover, this response was specific against P. salmonis antigens, since no cross reaction was detected with Renibacterium salmoninarum and Yersinia ruckeri. The vaccinated and control fish were challenged 60 days after the second dose of DNA with 2.5 x 10(7) P. salmonis corresponding to 7.5 times the LD50. At 30 days post-challenge, 100% mortality was obtained with the control fish while 20% of the vaccinated animals survived. All surviving fish exhibited a lower bacterial load in the kidney than control fish. The expression library was also tested in Balb/c mice and it was found that the humoral immune response was specific to P. salmonis and it was dependent on the amount of DNA injected.

Electrophoretic analysis of ITS from Piscirickettsia salmonis Chilean isolates.

Piscirickettsia salmonis is the most important pathogen in salmonid mariculture in Chile. Since it was reported numerous piscirickettsiosis outbreaks have occurred differing in virulence and mortality. Genetic variability of P. salmonis isolates has been suggested as one factor to explain this. However until now isolates obtained from outbreaks have not been analyzed. Knowledge of genetic variability of P. salmonis is very limited and also a useful screening method for genetic variations in isolates without sequencing is not available. Here we report an electrophoretic analysis of internal transcribed spacer region (ITS) of eleven P. salmonis isolates obtained from different salmon species and places in southern Chile. When PCR products were submitted to polyacrylamide gel electrophoresis (PAGE) a characteristic electrophoretic pattern was observed, distinguishable from ITS of other bacteria, including fish pathogens. Even though this pattern is conserved in all isolates, a difference in ITS electrophoretic mobility was observed, determining clearly two groups: ITS with higher or with lower electrophoretic mobility, including LF-89 and EM-90 isolates, respectively. A higher ITS sequence homology inside each group was shown by heteroduplex mobility assay (HMA). Our results show that genetic variability between Chilean P. salmonis isolates allows the differentiation of two groups with similar behavior observed previously when six P. salmonis isolates from three geographic origins were analyzed by 16S, 23S and ITS sequencing. PAGE analysis of ITS and HMA could be a basis to develop an assay for screening genetic variability between P. salmonis isolates.

Development and application of a nested PCR to monitor brood stock salmonid ovarian fluid and spleen for detection of the fish pathogen Flavobacterium psychrophilum.

AIMS: To develop a nested PCR to detect Flavobacterium psychrophilum based on the intergenic spacer region 16S-23S rRNA and in 16S rRNA for analysis of brood stock salmonid fish samples. METHODS AND RESULTS: The sensitivity and specificity of the test was evaluated using pure cultures, spiked and naturally contaminated samples. Samples were internal organs (spleen and kidney), eggs and ovarian fluid from rainbow trout and coho salmon from European fish farms (France, Spain). This nested PCR was more specific and sensitive that the nested PCR based on 16S rRNA sequences primers only. The detection limit of this PCR assay was one bacterium per PCR tube corresponding to 10 bacteria/mg of spleen and 5 bacteria/ml from ovarian fluid. Analysis of mixed ovarian fluid samples from reproductive salmonids in various French hatcheries demonstrated that 69% of hatcheries were contaminated with Fl. psychrophilum. The analysis of individual samples demonstrated that 39% of rainbow trout (Oncorhynchus mykiss) and 62.5% of coho salmon (O. kisutch) samples were contaminated. CONCLUSIONS: The results demonstrated a very sensitive and specific detection of this fish pathogen and that most of the female rainbow trout and coho salmon breeders analysed carry Fl. psychrophilum in the ovarian fluid. SIGNIFICANCE AND IMPACT OF THE STUDY: The understanding of Fl. psychrophilum dissemination and transmission and the detection of asymptomatic carriers is important for the development of free breeders stock and for significantly decreasing Flavobacteriose.

Antigenic characterization of the fish pathogen Flavobacterium psychrophilum.

Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrotroph Flavobacterium psychrophilum is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic diseases that heavily impact salmonids. Consequently, two verified but geographically diverse isolates were characterized phenotypically and biochemically. A facile typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates. F. psychrophilum was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low-molecular-mass carbohydrate antigen as well as several protein antigens. Surface-exposed antigens were visualized by a combination of immunoflourescence microscopy, immunogold transmission, and thin-section electron microscopy and were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTA-polymyxin B agarose beads, and by extrinsic labeling of amines with sulfo-N-hydoxysuccinimide-biotin and glycosyl groups with biotin hydrazide. The predominant approximately 16 kDa antigen was identified as low-molecular-mass lipopolysaccharide (LPS), whereas high-molecular-mass LPS containing O antigen was not as prevalent on whole cells but was abundant in culture supernatants. Rainbow trout convalescent antisera recognized both molecular mass classes of LPS as well as a predominant approximately 20-kDa protein. This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed F. psychrophilum strains.

Coho salmon Oncorhynchus kisutch strain differences in disease resistance and non-specific immunity, following immersion challenges with Vibrio anguillarum.

Two strains of freshwater-reared coho salmon Oncorhynchus kisutch were compared for differences in the activity of selected non-specific immune factors before and after lethal and non-lethal immersion challenges with the marine bacterial pathogen Vibrio anguillarum (Vang). Two disease challenge experiments were performed. The first experimental challenge resulted in no mortality; however, significant strain and challenge treatment effects were detected at Day 16 post-challenge. Strain differences in plasma lysozyme activity were found in pre-challenge samples. The second challenge experiment compared the same strains of coho salmon following immersion challenges in different doses of Vang. The fish were sampled at Days 0, 2, 7, and 18 post-challenge and mortality, plasma lysozyme, and anterior kidney phagocyte respiratory burst activity were compared. There were significant strain differences in mortality in the high dose group. The more disease-resistant strain was found to have higher levels of plasma lysozyme and anterior kidney phagocyte respiratory burst activity. These strain differences were detected at various times in the lethal (high dose) and non-lethal challenge groups. There was a clear relationship between the enhanced survival of the more disease-resistant strain and a more sustained, elevated non-specific immune response following the experimental disease challenges. The results of this study suggest that the basis for strain differences in innate disease resistance is related to the ability of the fish to respond quickly to the initial infection and to maintain the response until the infection is quelled.

A Piscirickettsia salmonis-like bacterium associated with mortality of white seabass Atractoscion nobilis.

Mortality among hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was associated with infections by a Piscirickettsia salmonis-like organism (WSPSLO). Infected fish had no consistent external signs other than pale gills, lethargy and impaired swimming behavior. Internally, the kidney and spleen were enlarged, and some fish had livers with multiple pale foci. Smears from infected kidney, liver, and spleen stained with Wright-Giemsa had intracytoplasmic coccoid organisms, often in pairs, that ranged in size from 0.5 to 1.0 microm. Microscopic lesions included multifocal hepatic, renal, and splenic necrosis, and intralesional macrophages often contained the WSPSLO. The bacterium was isolated from infected fish on cell lines of salmonid (CHSE-214) and white seabass (WSBK) origin. The WSPSLO induced plaque formation and destroyed the cell monolayers within 10 to 14 d incubation at temperatures of 15 and 20 degrees C. The bacterium retained infectivity for cell lines up to 14 d at 4 and 13 degrees C, up to 7 d at 20 degrees C, but it was inactivated at 37 and 56 degrees C within 24 and 1 h, respectively. Freezing at -20 degrees C reduced infectivity by 100-fold. Dehydration and resuspension in distilled water completely inactivated the bacterium. In contrast, the WSPSLO retained nearly all of its infectivity for CHSE-214 cells following a 72 h period in seawater at 20 degrees C. Polyclonal rabbit antibodies made to the WSPSLO reacted specifically in indirect fluorescent antibody tests (IFAT) with the bacterium in cell cultures and smears from infected fish tissues. Tissue smears from infected salmon or CHSE-214 cells with P. salmonis reacted weakly with the anti-WSPSLO serum. Conversely, polyclonal anti-P. salmonis serum produced a weakly positive reaction with the WSPSLO from infected CHSE-214 cells. The WSPSLO as propagated in CHSE-214 cells was highly virulent for juvenile coho salmon Oncorhynchus kisutch, inducing 80% mortality within 10 d of intraperitoneal injection of 10(2.5)-50% tissue culture infectious doses per fish. We conclude that the bacterium from white seabass possesses antigenic differences from P. salmonis yet possesses virulence for salmon equal to known strains of P. salmonis.

Development of a nested polymerase chain reaction for amplification of a sequence of the p57 gene of Renibacterium salmoninarum that provides a highly sensitive method for detection of the bacterium in salmonid kidney.

Nucleic acid-based assays have shown promise for diagnosing Renibacterium salmoninarum in tissues and body fluids of salmonids. Development of a nested polymerase chain reaction (PCR) method to detect a 320 bp DNA segment of the gene encoding the p57 protein of R. salmoninarum is described. Whereas a conventional PCR for a 383 bp segment of the p57 gene reliably detected 1000 R. salmoninarum cells per reaction in kidney tissue, the nested PCR detected as few as 10 R. salmoninarum per reaction in kidney tissue. Two DNA extraction methods for the nested PCR were compared and the correlation between replicate samples was generally higher in samples extracted by the QIAamp system compared with those extracted by the phenol/chloroform method. The specificity of the nested PCR was confirmed by testing DNA extracts of common bacterial fish pathogens and a panel of bacterial species reported to cause false-positive reactions in the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for R. salmoninarum. Kidney samples from 74 naturally infected chinook salmon were examined by the nested PCR, the ELISA, and the FAT, and the detected prevalences of R. salmoninarum were 61, 47, and 43%, respectively.

Molecular analysis of an A-protein secretion mutant of Aeromonas salmonicida reveals a surface layer-specific protein secretion pathway.

The Aeromonas salmonicida Tn5 mutant, A449-TM1, is unable to secrete the surface layer protein (A-protein) through the outer membrane. Immunogold labeling of thin sections of A449-TM1, with polyclonal antisera against the A-protein, showed the accumulation of large quantities of A-protein in an enlarged periplasm. The majority of the labeled A-protein could be seen at the poles of the cells. The ability of A449-TM1 to secrete other extracellular proteins such as hemolysin and protease was not impaired by the Tn5 insertion, which indicates that the mutation in A449-TM1 interferes with a secretion pathway specifically for the translocation of the A-protein through the outer membrane. The mutant, A449-TM1, was shown to be avirulent for fish. A cosmid clone from a gene library of A449-TM1, which contains the Tn5 insertion from the chromosome, was used to identify a 1.4 kb SaII/ClaI fragment from immediately adjacent to the Tn5 insertion. This fragment was used to identify and clone a 4 kb HindIII fragment from a chromosomal DNA digest from the wild-type strain, A449. DNA sequence analysis of this clone identified an open reading frame (ORF) of 1656 bp. The deduced product of this ORF showed sequence similarity to a family of ATP-binding secretion proteins, but appeared to be phylogenetically distinct from these proteins, consistent with its participation in a secretory pathway specific for surface layer protein.

An outbreak of myxobacterial disease in coho salmon (Oncorhynchus kisutch) reared in a Maine estuary.

An epizootic of a myxobacterial infection in coho salmon (Oncorhynchus kisutch) was responsible for the death of 50,000 fish, 30% of the population. Cartiage in the nose, mouth and lower jaw was eroded, and yellow sheets of bacterial growth were observed in the mouth, pharynx and pneumatic duct. The severity of the disease increased with increasing water temperature. Pathogenicity trials were inconclusive; only two of 18 experimentally infected fish succumbed to the disease. However, the lesions, and the absence of other known pathogens suggests the myxobacterium was responsible.