Characterization of the rRNA locus of Pfiesteria piscicida and development of standard and quantitative PCR-based detection assays targeted to the nontranscribed spacer

Pfiesteria piscicida is a heterotrophic dinoflagellate widely distributed along the middle Atlantic shore of the United States and associated with fish kills in the Neuse River (North Carolina) and the Chesapeake Bay (Maryland and Virginia). We constructed a genomic DNA library from clonally cultured P. piscicida and characterized the nontranscribed spacer (NTS), small subunit, internal transcribed spacer 1 (ITS1), 5.8S region, ITS2, and large subunit of the rRNA gene cluster. Based on the P. piscicida ribosomal DNA sequence, we developed a PCR-based detection assay that targets the NTS. The assay specificity was assessed by testing clonal P. piscicida and Pfiesteria shumwayae, 35 additional dinoflagellate species, and algal prey (Rhodomonas sp.). Only P. piscicida and nine presumptive P. piscicida isolates tested positive. All PCR-positive products yielded identical sequences for P. piscicida, suggesting that the PCR-based assay is species specific. The assay can detect a single P. piscicida zoospore in 1 ml of water, 10 resting cysts in 1 g of sediment, or 10 fg of P. piscicida DNA in 1 micro g of heterologous DNA. An internal standard for the PCR assay was constructed to identify potential false-negative results in testing of environmental sediment and water samples and as a competitor for the development of a quantitative competitive PCR assay format. The specificities of both qualitative and quantitative PCR assay formats were validated with >200 environmental samples, and the assays provide simple, rapid, and accurate methods for the assessment of P. piscicida in water and sediments.     

Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 microM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 microM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.     

Identification and characterisation of the fur genes in Photobacterium damselae ssp. piscicida and ssp. damselae

The gene encoding the ferric uptake regulator protein (fur gene) of the fish pathogenic bacterium Photobacterium damselae ssp. piscicida Strain D121 was partially amplified using degenerate oligonucleotides. Complete sequencing of the fur gene and neighbouring DNA was accomplished by primer walking. An open reading frame of 447 bp, coding for a protein of 148 amino acids, and with high homology to previously described Fur proteins, was identified. The fur gene of P. damselae ssp. damselae ATCC 35083 was subsequently amplified by PCR with specific primers and its sequence determined, showing a 99.3% similarity to the P. damselae ssp. piscicida fur gene. The P. damselae fur gene was able to complement the fur mutation of Escherichia coli Strain H1681 in an iron-dependent fashion.     

Simple and rapid detection of Photobacterium damselae ssp. piscicida by a PCR technique and plating method

AIMS: To detect Photobacterium damselae ssp. piscicida using the PCR technique and plating method. METHODS AND RESULTS: Two strains of P. damselae ssp. piscicida were isolated from cultured cobia (Rachycentron canadum) at two different fish farms in Taiwan. A pair of primers was designed to detect the capsular polysaccharide gene of P. damselae ssp. piscicida by PCR. Reference strains of different genus and different clinical strains were used for this study. The expected product (410 bp) was obtained from both P. damselae ssp. piscicida and P. damselae ssp. damselae, and they were differentiated by culturing on thiosulphate citrate bile salts-sucrose agar (TCBS-1). Photobacterium damselae ssp. damselae grew on TCBS-1 producing green colonies whereas P. damselae ssp. piscicida did not grow. CONCLUSIONS: The methods used are cost and labour effective when compared with the other methods and commercially available kits. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides an integrated set of methods to identify the species P. damselae and to differentiate P. damselae ssp. piscicida from P. damselae ssp. damselae.     

Multiplex PCR assay for ureC and 16S rRNA genes clearly discriminates between both subspecies of Photobacterium damselae

A multiplex-PCR approach, employing 2 primer pairs directed to internal regions of the 16S rRNA and ureC genes, was utilized to analyze a collection of Photobacterium damselae strains, including 25 isolates of subspecies piscicida and 15 isolates of subspecies damselae. With this procedure, all the P. damselae subsp. damselae strains yielded 2 amplification products, one of 267 bp and the other of 448 bp, corresponding to internal fragments of the 16S rRNA and ureC genes, respectively. However, P. damselae subsp. piscicida isolates only showed the PCR product of 267 bp (16S rRNA fragment), indicating the absence of the urease gene in its genome. We have constructed a DNA probe directed to an internal region of the ureC gene, and corroborated by dot blot hybridization that the P. damselae subsp. piscicida lacks this gene, whereas it is present in the subspecies damselae. This constitutes the first successful discrimination between both subspecies using a PCR procedure, which could become a useful tool for diagnosis of pasteurellosis in the field. In addition, since these 2 subspecies have been shown to share nearly the same rrn operon sequence, our results provided evidence that one of the steps in the P. damselae speciation proccess included gain/loss events associated with the ure operon.     

16S rRNA gene sequence analysis of Photobacterium damselae and nested PCR method for rapid detection of the causative agent of fish pasteurellosis.

The causative agent of fish pasteurellosis, the organism formerly known as Pasteurella piscicida, has been reclassified as Photobacterium damselae subsp. piscicida on the basis of 16S rRNA gene sequence comparisons and chromosomal DNA-DNA hybridization data; thus, this organism belongs to the same species as Photobacterium damselae subsp. damselae (formerly Vibrio damselae). Since reassignment of P. damselae subsp. piscicida was based on only two strains, one objective of the present work was to confirm the taxonomic position of this fish pathogen by sequencing the 16S rRNA genes of 26 strains having different geographic and host origins. In addition, a nested PCR protocol for detection of P. damselae based on 16S rRNA was developed. This PCR protocol was validated by testing 35 target and 24 nontarget pure cultures, and the detection limits obtained ranged from 1 pg to 10 fg of DNA (200 to 20 cells). A similar level of sensitivity was observed when the PCR protocol was applied to fish tissues spiked with bacteria. The PCR approach described in this paper allows detection of the pathogen in mixed plate cultures obtained from asymptomatic fish suspected to be carriers of P. damselae subsp. piscicida, in which growth of this bacterium cannot be visualized. Our results indicate that the selective primers which we designed represent a powerful tool for sensitive and specific detection of fish pasteurellosis.     

一种利用λRed重组技术在杀鱼爱德华氏菌基因组添加标签的方法

在杀鱼爱德华氏菌病原学研究中,在动物中制备杀鱼爱德华氏菌蛋白抗体耗时长,且获得的多克隆或多肽抗体在宿主细胞中特异性差,背景信号强。为解决这一问题,对在大肠杆菌(Escherichia coli)和沙门氏菌(Salmonella)中建立起来的λRed基因编辑方法进行调整和优化,建立了在杀鱼爱德华氏菌(Edwardsiella piscicida)基因组基因上添加HA标签序列的方法,为使用标签抗体研究杀鱼爱德华氏菌基因功能提供便利。λRed重组系统利用同源线性DNA片段与基因组DNA进行重组。即以质粒pSU315为模板,在引物上引入目的基因的特异性序列,扩增FRT序列和抗生素抗性基因;以获得的PCR产物转化携带pKD46的杀鱼爱德华氏菌,在pKD46表达的λ噬菌体的3个重组蛋白(Exo、Beta和Gam)作用下, PCR产物与杀鱼爱德华氏菌基因组发生同源重组,获得引入了抗生素抗性的靶基因缺失或靶基因携带标签序列的菌株;接着利用pKD46的温敏型特性,消除引入的pKD46;最后向杀鱼爱德华氏菌引入文章构建的表达Flp重组酶的质粒pKD46-flp,在FLP作用下,两个FRT位点之间发生重组,...     

MIXOTROPHY AND NITROGEN UPTAKE BY PFIESTERIA PISCICIDA (DINOPHYCEAE)

The nutritional versatility of dinoflagellates is a complicating factor in identifying potential links between nutrient enrichment and the proliferation of harmful algal blooms. For example, although dinoflagellates associated with harmful algal blooms (e.g. red tides) are generally considered to be phototrophic and use inorganic nutrients such as nitrate or phosphate, many of these species also have pronounced heterotrophic capabilities either as osmotrophs or phagotrophs. Recently, the widespread occurrence of the heterotrophic toxic dinoflagellate, Pfiesteria piscicida Steidinger et Burkholder, has been documented in turbid estuarine waters. Pfiesteria piscicida has a relatively proficient grazing ability, but also has an ability to function as a phototroph by acquiring chloroplasts from algal prey, a process termed kleptoplastidy. We tested the ability of kleptoplastidic P. piscicida to take up ¹⁵N-labeled NH     , NO     , urea, or glutamate. The photosynthetic activity of these cultures was verified, in part, by use of the fluorochrome, primulin, which indicated a positive relationship between photosynthetic starch production and growth irradiance. All four N substrates were taken up by P. piscicida , and the highest uptake rates were in the range cited for phytoplankton and were similar to N uptake estimates for phagotrophic P. piscicida . The demonstration of direct nutrient acquisition by kleptoplastidic P. piscicida suggests that the response of the dinoflagellate to nutrient enrichment is complex, and that the specific pathway of nutrient stimulation (e.g. indirect stimulation through enhancement of phytoplankton prey abundance vs. direct stimulation by saprotrophic nutrient uptake) may depend on P. piscicida 's nutritional state (phagotrophy vs. phototrophy).     

Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.     

Evaluation of different DNA-based fingerprinting methods for typing Photobacterium damselae ssp. piscicida

This study evaluates the effectiveness of three different molecular techniques, repetitive extragenic palindromic PCR (REP-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and the random amplified polymorphic DNA (RAPD-PCR) for rapid typing of Photobacterium damselae ssp. piscicida strains isolated from different species of marine fish and geographic areas. The results obtained by the three methods showed that RAPD and ERIC-PCR were more discriminative for suitable rapid typing of Ph. damselae ssp. piscicida than REP-PCR. The analysis of DNA banding patterns generated by both molecular methods (RAPD and ERIC-PCR) clearly separated the strains into two main groups that strongly correlated with their geographic origin. Moreover, the REP-PCR analysis was less reproducible than the RAPD and ERIC-PCR methods and does not allow the establishment of genetic groups. RAPD and ERIC-PCR constitute valuable tools for molecular typing of Ph. damselae ssp. piscicida strains, which can be used in epidemiological studies of photobacteriosis infections.     

THE PHYLOGENETIC RELATIONSHIP OF PFIESTERIA PISCICIDA, CRYPTOPERIDINIOPSOID SP. AMYLOODINOUM OCELLATUM AND A PFIESTERIA-LIKE DINOFLAGELLATE TO OTHER DINOFLAGELLATES AND APICOMPLEXANS

The taxonomic relationship between heterotrophic and parasitic dinoflagellates has not been studied extensively at the molecular level. In order to investigate these taxonomic relationships, we sequenced the small subunit (SSU) ribosomal RNA gene of Pfiesteria piscicida (Steidinger et Burkholder), a Pfiesteria -like dinoflagellate, Cryptoperidiniopsoid sp., and Amyloodinium ocellatum (Brown) and submitted those sequences to GenBank. Pfiesteria piscicida and Cryptoperidiniopsoid sp. are heterotrophic dinoflagellates, purportedly pathogenic to fish, and A. ocellatum, a major fish pathogen, has caused extensive economic losses in both the aquarium and aquaculture industries. The pathogenicity of the Pfiesteria -like dinoflagellate is unknown at this time, but its growth characteristics and in vitro food preferences are similar to those of P. piscicda. The SSU sequences of these species were aligned with the other full-length dinoflagellate sequences, as well as those of representative apicomplexans and Perkinsus species, the groups most closely related to dinoflagellates. Phylogenetic analyses indicate that Cryptoperidiniopsoid sp., P. piscicida, and the Pfiesteria -like dinoflagellate are closely related and group into the class Blastodiniphyceae, as does A. ocellatum. None of the species examined were closely related to the apicomplexans or to Perkinsus marinus, the parasite that causes "Dermo disease" in oysters. The overall phylogenetic analyses largely supported the current class and subclass groupings within the dinoflagellates.     

Photobacterium damselae ssp. piscicida: detection by direct amplification of 16S rRNA gene sequences and genotypic variation as determined by amplified fragment length polymorphism (AFLP)

A PCR protocol for the rapid diagnosis of fish 'pasteurellosis' based on 16S rRNA gene sequences was developed. The procedure combines low annealing temperature that detects low titers of Photobacterium damselae but also related species, and high annealing temperature for the specific identification of P. damselae directly from infected fish. The PCR protocol was validated on 19 piscine isolates of P. damselae ssp. piscicida from different geographic regions (Japan, Italy, Spain, Greece and Israel), on spontaneously infected sea bream Sparus aurata and sea bass Dicentrarchus labrax, and on closely related American Type Culture Collection (ATCC) reference strains. PCR using high annealing temperature (64 degrees C) discriminated between P. damselae and closely related reference strains, including P. histaminum. Sixteen isolates of P. damselae ssp. piscicida, 2 P. damselae ssp. piscicida reference strains and 1 P. damselae ssp. damselae reference strain were subjected to Amplified Fragment Length Polymorphism (AFLP) analysis, and a similarity matrix was produced. Accordingly, the Japanese isolates of P. damselae ssp. piscicida were distinguished from the Mediterranean/European isolates at a cut-off value of 83% similarity. A further subclustering at a cut-off value of 97% allowed discrimination between the Israeli P. damselae ssp. piscicida isolates and the other Mediterranean/European isolates. The combination of PCR direct amplification and AFLP provides a 2-step procedure, where P. damselae is rapidly identified at genus level on the basis of its 16S rRNA gene sequence and then grouped into distinct clusters on the basis of AFLP polymorphisms. The first step of direct amplification is highly sensitive and has immediate practical consequences, offering fish farmers a rapid diagnosis, while the AFLP is more specific and detects intraspecific variation which, in our study, also reflected geographic correspondence. Because of its superior discriminative properties, AFLP can be an important tool for epidemiological and taxonomic studies of this highly homogeneous genus.     

Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.     

Assessment of a magnetic bead-EIA based kit for rapid diagnosis of fish pasteurellosis.

The accuracy of the magnetic beads-EIA based BIONOR AQUAEIA-Pp kit for the rapid diagnosis of pasteurellosis was evaluated. The kit reacted with all the Photobacterium damselae subsp. piscicida strains included in this study, with a detection limit of 10(4) bacteria/ml. However, non-specific reactions were observed with isolates of Ph. damselae subsp. damselae or Ph. histaminum when the bacterial concentration was high (10(9)-10(10) bacteria/ml). Similar findings in specificity and sensitivity were observed when the kit was applied to experimentally infected fish tissues. However, since those bacterial species are not usually found in the fish species susceptible to pasteurellosis, the AQUAEIA kit appears applicable for a rapid screening of the disease. In addition, when the kit was utilized to analyze cultured populations of seabream, it allowed the detection of the pathogen, not only in individuals affected by the disease, but also in asymptomatic carrier fish. Furthermore, the positive detection of Ph. damselae subsp. piscicida in broodstock gonads, seminal, and ovaric fluids, and also in eggs indicated the possibility of vertical transmission of pasteurellosis.     

Molecular diversity of Photobacterium damselae ssp. piscicida from cultured amberjacks (Seriola spp.) in Japan by pulsed-field gel electrophoresis and plasmid profiles

AIMS: This study deals with a pulsed-field gel electrophoresis (PFGE) for discriminating between the genetic variants of Photobacterium damselae ssp. piscicida, and characterizing of Japanese field isolates by PFGE together with plasmid profiles and antimicrobial resistances. METHODS AND RESULTS: A total of 74 field isolates from cultured Japanese amberjacks were used for PFGE. SmaI and NotI enabled to clearly differentiate strains and we obtained 24 of combined PFGE profiles which were distinct from those of classical Japanese and USA reference strains, and classified them into three groups (Ia-Ic). By plasmid size, we could classify these field isolates into three plasmid types, pA-pC. The predominant PFGE-type Ia was closely associated with plasmid-type pA, and Ib showed a moderate association with pB. Ic was closely associated with pC, and multiresistant isolates were not observed in this type. Whole-genomic variations were also observed between isolates having identical detection areas, fish species and detection-date by PFGE. CONCLUSION: Molecular diversity of P. damselae ssp. piscicida could be detected by PFGE, and some relations among the PFGE-type, plasmid-type and antimicrobial resistances were observed in Japanese field isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicated that some genetic transition might have occurred in P. damselae ssp. piscicida around the Japanese seas, and PFGE can be a valuable tool for the epidemiological study of this highly homogeneous subspecies.     

Comparative culture and toxicity studies between the toxic dinoflagellate Pfiesteria piscicida and a morphologically similar cryptoperidiniopsoid dinoflagellate

A series of fish bioassays using cultures of the toxic dinoflagellate, Pfiesteria piscicida and a cryptoperidiniopsoid dinoflagellate indicated various degrees of toxicity for Pfiesteria piscicida and no toxicity by the cryptoperidiniopsoid. P. piscicida maintained toxicity in the presence of live fish, and this toxicity was perpetuated following a series of inoculations to other culture vessels. Differences in the onset and magnitude of the fish deaths occurred, requiring 16 days for the initial fish death when using P. piscicida from a culture that had previously been maintained on algal cells, to kills within hours when using a culture that had recently (previous day) killed fish. Autopsies of moribund fish from the test and control fish bioassays indicated a general lack of bacterial infection, which ensued following death of other autopsied fish. Moreover, bacterial comparisons of waters in the fish bioassay and control fish cultures indicated that similar bacterial concentrations were present. Neither oxygen or ammonia levels were determined to be factors in the fish death. Life stages of a cryptoperidiniopsoid dinoflagellate from Virginia estuaries were also identified, including motile zoospore, gametes, planozygote, amoebae, and cyst stages. The cryptoperidiniopsioid did not initiate fish deaths in bioassays conducted over a 14-week period at zoospore concentrations of ca. 700-800 cells ml(-1). Elemental X-ray analysis of the scales from cysts of this dinoflagellate and P. piscicida indicate that they both contain silicon. Overall, the data from this study demonstrate that the cryptoperidiniopsoid possesses several similar life stages and feeding patterns as P. piscicida, but was not toxic to fish.     

Construction of a safe, stable, efficacious vaccine against Photobacterium damselae ssp. piscicida

Vaccination with bacterial auxotrophs, particularly those with an interruption in the common pathway of aromatic amino-acid biosynthesis, known as the shikimate pathway, has been shown to be effective in the prevention of a variety of bacterial diseases. In order to evaluate this approach to vaccine development in the important marine pathogen Photobacterium damselae subsp. piscicida, the aroA gene of the shikimate pathway was identified from a P. damselae subsp. piscicida genomic library by complementation in an aroA mutant of Escherichia coli. The complementing plasmid was isolated and the nucleotide sequence of the P. damselae subsp. piscicida genomic insert was determined. Subsequent analysis of the DNA-sequence data demonstrated that the identified plasmid contained 3464 bp of P. damselae subsp. piscicida DNA, including the complete aroA gene. The sequence data was used to delete a 144 bp MscI fragment, and the kanamycin resistance gene (kan) from transposon Tn903 was ligated into the MscI site. This delta(aro)A::kan construct was sub-cloned into a suicide plasmid and transferred to a wild-type P. damselae subsp. piscicida by conjugation and allelic exchange. One selected mutant, LSU-P2, was confirmed phenotypically to require supplementation with aromatic metabolites for growth in minimal media, and was confirmed genotypically by PCR and DNA sequencing. Further, LSU-P2 was demonstrated to be avirulent in hybrid striped bass and to provide significant protection against disease following challenge with the wild-type strain.     

Iron uptake by Pasteurella piscicida and its role in pathogenicity for fish.

We evaluated the iron uptake mechanisms in Pasteurella piscicida strains as well as the effect of iron overload on the virulence of these strains for fish. With this aim, the capacity of the strains to obtain iron from transferrin and heme compounds as well as their ability to overcome the inhibitory activity of fish serum was analyzed. All the P. piscicida strains grew in the presence of the iron chelator ethylene-diamine-di (O-hydroxyphenyl acetic acid) or of human transferrin, which was used by a siderophore-mediated mechanism. The chemical tests and cross-feeding assays showed that P. piscicida produced a siderophore which was neither a phenolate nor a hydroxamate. Cross-feeding assays as well as preliminary chromatographic analysis suggest that this siderophore may be chemically related to multocidin. All the P. piscicida isolates utilized hemin and hemoglobin as an iron source, since the virulence of the strains increased when the fish were preinoculated with these compounds. This effect was stronger in the avirulent strains (50% lethal dose was reduced by 4 logs when fish were pretreated with hemin or hemoglobin). Only the pathogenic P. piscicida isolates were resistant to the bactericidal action of the fresh fish serum. The nonpathogenic strains grew in fish serum only when it was heat-inactivated or when it was supplemented with ferric ammonium citrate, hemin, or hemoglobin. In all the strains, at least three iron-regulated outer membrane proteins (IROMPs) (105, 118, and 145 kDa) were increased when the strains were cultured in iron-restricted medium.(ABSTRACT TRUNCATED AT 250 WORDS)