Comparative in vitro studies on virulent and avirulent strains of Cryptobia salmositica (Sarcomastigophora: Kinetoplastida)

Abstract. The optimum temperature for in vitro multiplication of Cryptobia salmositica was 10°C. The avirulent strain multiplied more rapidly than the virulent strain. The haemolytic components, lytic component (LC) and immune complex-forming component (ICC), were secreted by the two strains into the culture medium and were detectable from one week post-inoculation. The haemolytic activity in the supernatant increased with increasing parasite numbers in both strains. Although cultures of the avirulent strain had higher parasite numbers than those of the virulent strain, the haemolytic activity was significantly lower than that of the virulent strain. Antiserum against ICC was produced in rabbit by immunization with ICC-coated rainbow trout red blood cells.     

Inhibitory effects of a monoclonal antibody (MAb-001) on in vitro oxygen consumption and multiplication of the pathogenic haemoflagellate, Cryptobia salmositica Katz

A monoclonal antibody (MAb-001), against a surface glycoprotein on Cryptobia salmositica inhibited the multiplication and oxygen consumption of both virulent and avirulent strains of the parasite. The classical cysteine proteinase inhibitor (E-64) and a cysteine proteinase activator (EDTA) affected the in vitro multiplication of C. salmositica . Concentrations of E-64 higher than 10 μ M reduced the multiplication of C. salmositica while 5 m M of EDTA enhanced its multiplication. We propose that the cysteine proteinase is an important metabolic enzyme in C. salmositica and that binding of MAb-001 to the enzyme inhibited parasite multiplication and reduced oxygen consumption.     

Conjugation of isometamidium chloride to antibodies and the use of the conjugate against the haemoflagellate, Cryptobia salmositica Katz, 1951: an immuno-chemotherapeutic strategy

The trypanocidal drug isometamidium chloride (Samorin) was conjugated to polyclonal and monoclonal antibodies produced against the pathogenic haemoflagellate Cryptobia salmositica . Under in vitro conditions the unconjugated drug normally accumulates rapidly in the kinetoplast in the parasite; however, once it was conjugated to antibodies (either polyclonal or monoclonal) it was found throughout the parasite. Isometamidium conjugated to polyclonal antibodies lysed C. salmositica under in vitro conditions, but parasites were not agglutinated. In contrast, isometamidium conjugated to monoclonal antibodies (against a 200 kDa surface membrane glycoprotein) did not lyse C. salmositica , but parasites were agglutinated. Because of the low efficacy of the monoclonal conjugate against the parasite in vitro , its cryptobiocidal effect was not evaluated further. The infectivity of C. salmositica (incubated either in culture medium or whole blood) was reduced in fish after in vitro exposure to isometamidium conjugated to polyclonal antibodies. Parasitaemias were reduced in infected chinook salmon, Oncorhynchus tshawytscha, after treatment with isometamidium conjugated to polyclonal antibodies.     

Adherence, haemagglutination and cell surface characteristics of motile aeromonads virulent for fish

Abstract. Motile Aeromonas spp. virulent for fish were studied with regard to their adhesion profile. Electron microscopy demonstrated the presence of fimbriae (pili) on Aeromonas cells regardless of virulence potential. The results show no significant correlations between ability to haemagglutinate, yeast cell co-agglutination and virulence. All strains expressed mannose-sensitive haemagglutinin activity against guinea pig erythrocytes. Using four types of red blood cells no characteristic haemagglutinin pattern related to virulence could be discerned. Expression of surface haemagglutin(s) on Aeromonas hydrophila appears to be medium dependent; strains grown in liquid media demonstrated enhanced haemagglutination activity. Both virulent and avirulent strains had in vitro epithelial cell adhesive capabilities. Cell surface characteristics measured by agglutination in acriflavine and stability after boiling indicated that most virulent strains agglutinated in the presence of acriflavine, but not all sedimented after boiling. The ability of 10 selected strains of A hydrophila to grow in normal pooled catfish serum was determined. Only 17% of the virulence variations can be explained by their sensitivity to serum.     

Interaction of the fish pathogen Aeromonas hydrophila with tilapia, Oreochromis aureus (Steindachner), phagocytes

Abstract. Interaction of Aeromonas hydrophila and tilapia, Oreochromis aureus (Steindachner), phagocytes was studied in vitro. All virulent and avirulent strains of A. hydrophila tested could multiply in non-activated and Freund's complete adjuvant activated phagocytes. Activated phagocytes increased the uptake of bacteria into cells, and the rates of intracellular replication for these bacteria were faster than in non-activated phagocytes. Among the A. hydrophila strains examined, virulent strain PPD134/91 replicated at the fastest rate inside phagocytic cells and produced cytopathic effect in the phagocytes in the shortest incubation time. Opsonized avirulent A. hydrophila were sensitive to phagocyte-mediated killing or unable to grow in phagocytes. Serum components and phagocytes may together prevent the growth of avirulent A. hydrophila in fish. The release of extracellular oxygen radicals during phagocytosis was examined using chemiluminescence assay (CL). Virulent strains induced CL responses but avirulent strains did not. This suggests that the virulent strains interacted with the phagocytes somewhat differently from the avirulent strains.     

Aerococcus viridans expression of Cpn60 is associated with virulence during infection of the American lobster, Homarus americanus Milne Edwards

The Gram-positive bacterium Aerococcus viridans var. homari is a well-documented causative agent of the lethal systemic disease gaffkemia in both the American lobster, Homarus americanus, and the European lobster, Homarus gammarus. Previous phenotypic characterization has been unsuccessful at differentiating avirulent from virulent strains without performing lethal animal infection trials. Recent genetic characterization of A. viridans strains through 16S rRNA sequencing and random amplification of polymorphic DNA fingerprinting has revealed the presence of two subtypes. However, subtype 1 contains both virulent and avirulent strains which are genetically identical. The purpose of this study was to determine the proteomic mediators of virulence in A. viridans. Quantitative proteomic mapping of these two strains has revealed 29 differentially expressed protein spots, seven of which are only expressed in the virulent strain and could act as virulence factors. One protein, chaperonin 60 (Cpn60), is uniquely expressed in the virulent strain and has been shown to act as a virulence factor in many other bacteria. The proteomic mapping strategy employed in this study is the first to show phenotypic differences between virulent and avirulent strains. Cpn60 expression represents a potentially useful tool for identifying the virulent strains of A. viridans in epidemiological studies.     

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non‐virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE‐214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non‐virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin‐D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non‐virulent strains were generally serum sensitive.     

Immunogenicity of vaccines from a virulent and an a virulent strain of Aeromonas salmonicida

Abstract. Injected vaccines consisting of formalin-killed cells and extracellular antigens prepared from a virulent and an avirulent strain of Aeromonas salmonicida were tested for their efficacy in protecting juvenile coho salmon. Oncorhychus kisutch (Walbaum). Against experimental furunculosis following active and passive immunization. Sera used in the passive immunization experiments were raised in subadult coho salmon and in rabbits. Results indicated that the avirulent strain was inferior to the virulent strain in its immunogenicity for coho salmon. Thus, even though avirulent cells possessed at least one immunogen–an immunogen that was show in passive immunization experiments to be well recognized by rabbits–the immunogen was only inefficiently protection when vaccinated with avirulent cells. Furher, extracellular antigens of the avirulent strain were not protective for coho salmon even though they elicited the production of anti- A. salmonicida agglutinins. In contrast, the killed cells and extracellular antigens of the virulent strain were both immunogenic in coho salmon. By passively immunizing coho salmon with rabbit sera raised against the virulent and the virulent strain produced at least two immunogens, only one of which was shared in common with the avirulent strain. The extra immunogen possessed by the virulent strain is presumably responsible for its superior immunogenicity in coho salmon; the immunogen had the properties of a protein and is believed to be the A-protein.     

Pathogenicity of Edwardsiella tarda to olive flounder, Paralichthys olivaceus (Temminck & Schlegel)

The LD50 and cytotoxic and enzymatic activities of both cells and extracellular products (ECPs) of eight Edwardsiella tarda strains were determined and their bacterial superoxide dismutase gene (sodB) and catalase gene (katB) were sequenced. Strains were also examined for their ability to resist the immune responses of olive flounder, Paralichthys olivaceus. LD50 values of strains (FSW910410, KE1, 2, 3, 4, 5 and 6) in olive flounder ranged between 10(2.5) and 10(5.3) cfu (colony forming units) per fish. Unlike the avirulent strain SU100 (LD50>or=10(7)), all pathogenic strains were able to survive in flounder serum and head kidney leucocytes (except for KE2). The virulent strains possessed type I sodB and katB, whereas SU100 had type II sodB but not katB. However, there was no difference between avirulent and virulent strains in haemolytic and cytotoxic activities. The results of this study demonstrated that the ability of E. tarda to resist complement activity and phagocytosis is conferred by its superoxide dismutase and catalase, which thus play an essential role in the pathogenicity of this bacterium. In addition genotyping of sodB and kat B proved to be a very useful tool to distinguish virulent from avirulent strains.     

A novel multiplex PCR method for detecting virulent strains of Vibrio alginolyticus

The bacterial strains obtained from various origins were tested with the novel primers targeting the collagenase gene, ompK gene and toxR gene to establish a multiplex polymerase chain reaction (PCR) method. These primers successfully recognized all virulent strains of Vibrio alginolyticus, but the avirulent strains were not recognized by the multiplex PCR because of lack of the collagenase and toxR genes. In a 50 μL multiplex PCR mixture, the lowest detection limit is 8.8 × 10² cells of virulent strains of V. alginolyticus. The multiplex PCR method was successfully developed to identify virulent strains of V. alginolyticus, and provides a rapid, sensitive, specific and reliable technology for diagnosing virulent strains of V. alginolyticus. Therefore, the novel multiplex PCR in the present paper can be useful for any laboratory working with vibriosis detection of aquatic animals.     

Comparative analysis of the phenotypic characteristics of high- and low-virulent strains of Edwardsiella tarda

Edwardsiella tarda is a causative agent of edwardsiellosis in freshwater and marine fish. Extracellular enzymic, haemolytic, hydrophobic and serum resistance activities, haemagglutination, autoagglutination and siderophores of high‐ and low‐ virulent E. tarda strains were examined. The results revealed different haemagglutination, autoagglutination, haemolytic, hydrophobic and serum resistance activities in different strains. Analysis of extracellular proteins (ECPs) and outer membrane proteins (OMPs) demonstrated several major, low molecular weight, virulent‐strain‐specific proteins, which could be virulence‐related. Based on the database search with MALDI‐TOF MS data, the closest homologies of the three protein bands Ed1, Ed2 and Ed3 were phosphotransferase enzyme family protein, nitrite reductase [NAD(P)H], large subunit and ATP‐dependent Lon protease, respectively. A comparison of pathogenicity of purified lipopolysaccharide (LPS) and lipid A from virulent and avirulent strains demonstrated that LPS was one of the virulence factors of the E. tarda isolates, and lipid A was a biologically active determinant of LPS.     

Development and characterization of rifampicin-resistant mutants from high virulent strains of Flavobacterium columnare

Flavobacterium columnare is divided into three genetic groups or genomovars, genomovar II being highly virulent for channel catfish. A modified live vaccine is currently available to prevent columnaris disease under the licensed name Aquavac‐Col^®. The strain of F. columnare used to generate the avirulent rifampicin‐resistant mutant used in Aquavac‐Col^® belonged to genomovar I, the less virulent group towards channel catfish. In this study, we describe the generation and characterization of rifampicin‐resistant mutants from genomovar II strains. A total of 13 new mutants were obtained, and eight of them (two from each parent strain) were genetically and phenotypically characterized. Highly conserved regions within the ribosomal operons were identical between parent and mutant strains. Genetic differences between mutants and their parent strains were revealed by amplified fragment length polymorphism (AFLP). Genetic changes were distinctive among different mutants. Analysis of the lipopolysaccharide (LPS) showed that while some mutants lacked a few molecular bands of the LPS, some exhibited the same LPS profiles as their parent strains. Comparison between immunogenic proteins from mutants and parents was carried out by immunoblot analysis and further confirmed the uniqueness of individual mutants. A complete set of rifampicin‐resistant mutants with different genetic and immunogenic properties from the highly virulent genomovar II has been created. These mutants may have the potential of becoming vaccine candidates against columnaris disease.     

Characterization of glycans in the developmental stages of Myxobolus cerebralis (Myxozoa), the causative agent of whirling disease

Glycans and sugar-binding molecules (lectins) form an interactive recognition system, which may enable parasitic organisms to adhere to host cells and migrate into target tissues. The aim of the present study was to analyse surface-associated glycans in the developmental stages of Myxobolus cerebralis (Hofer), the causative agent of whirling disease. A panel of biotin-labelled plant lectins was used to detect a broad spectrum of glycan motifs with high specificity. Binding sites were detected histochemically in the tissue sections of infected rainbow trout, Oncorhynchus mykiss (Walbaum), and infected Tubifex tubifex (Müller), and were characterized by light, fluorescence and transmission electron microscopy. With mannose-specific lectins [Lens culinaris agglutinin, Pisum sativum agglutinin, Canavalia ensiformis agglutinin (LCA, PSA, CanA)] mannose-containing glycans were detected in all the developmental stages and host tissues. No binding sites for galactose-specific lectins were present in M. cerebralis spores but reactivity with host tissues occurred. Diversity in glycans was detected by N-acetyl-D-galactosamine-specific lectins in sporoplasm cells of M. cerebralis and triactinomyxon spores. In the group of lectins with monosaccharide-specificity for N-acetyl-D-glucosamine (GlcNAc), the reactivity of Datura stramonium agglutinin (DSA), Lycopersicon esculentum agglutinin (LEA) and Solanum tuberosum agglutinin (STA) was restricted to polar capsules whereas Griffonia simplicifolia agglutinin II (GSA II) also bound to sporoplasm cells of stages in the fish host but not in those present in infected T. tubifex. Moreover, Triticum vulgaris (wheat germ) agglutinin (WGA) and succinylated WGA indicated the presence of N-acetyl-D-glucosamine polymers in polar capsules. No specificity for spores was observed concerning 'bisected'N-glycans and no reactivity in parasitic stages was observed with the fucose-binding lectin Ulex europaeus agglutinin (UEA) I, Sambucus nigra agglutinin (SNA) (specific for alpha2,6-sialylated glycans) and Maackia amurensis agglutinin (MAAI) (specific for alpha2,3-sialylated glycans). Arachis hypogaea (peanut) agglutinin (PNA), Erythrina cristagalli agglutinin (ECA), GSA I, Sophora japonica agglutinin (SJA), Dolichos biflorus agglutinin (DBA) and GSA II detected reactive sites solely confined to the developmental stages of M. cerebralis and were not reactive in the fish host. These parasite-specific glycans may play a role in the adhesion process of the parasite to fish epidermis prior to infection, but may provide protection to the host by activating the complement system, or stimulating an adaptive immune response as putative antigens.     

Aeromonas hydrophila B-haemolysin: purification and examination of its role in virulence in 0-group channel catfish, Ictalurus punctatus (Rafinesque)

Abstract. A single step procedure for purification of B-haemolysin from Aeromonas hydrophila is described which recovered 94% of the haemolytic activity with a sixfold increase in specific activity. Toxicity of haemolysin in 0-group channel catfish was 0–6 mg of purified protein per gram offish. Of six strains of A. hydrophila evaluated for differential toxin production and virulence in 0-group channel catfish, three B-haemolysin-deficient strains were virulent while three B-haemolysin-producing strains were avirulent.     

Biological characterization of a monoclonal antibody against a surface membrane antigen on Cryptobia salmositica Katz, 1951

A monoclonal antibody (IgG 1) (designated as MAb-001) was produced against the pathogenic haemoflagellate Cryptobia salmontica Katz. The antibody agglutinated live parasites under in vitro conditions. Live C. salmositica, incubated with MAb-001 at 10 °C, did not multiply and were dead within 4 weeks in culture. About 50% of juvenile rainbow trout, Oncorhynchus mykiss (Walbaum), inoculated intraperhoneally with C. salmositica, incubated in MAb-001 prior to inoculation, did not become infected, while in adult rainbow trout, the peak parasitaemia was reduced. These results indicate that MAb-001 is a protective monoclonal antibody and the antigen it recognizes is located on rhe surface membrane of C. salmositica. The antibody also inhibits multiplication and affects viability of the parasite under in vitro conditions.     

Carbohydrate and lectin interactions with Edwardsiella ictaluri and channel catfish, Ictalurus punctatus (Rafinesque), anterior kidney leucocytes and hepatocytes

Abstract. Seven isolates of Edwardsiella ictaluri were evaluated for channel catfish red blood cell and neutrophil agglutination properties and reactivity with lectins and carbohydrates. All isolates were agglutinated by lectin derived from Ricinus communis and the intensity of the agglutination reaction could be lessened or abrogated by premixing the lectin derived from Ricinus communis with β-D-galactose or α-L-fructose. Edwardsiella ictaluri isolates also sedimented much more rapidly when mixed with lectin derived from Ricinus communis than when mixed with other lectins. Premixing Ricinus communis with β-D-galactose had the greatest effect on slowing the rate of sedimentation. Two out of the seven E. ictaluri isolates were capable of agglutinating channel catfish red blood cells and anterior kidney neutrophils, and this agglutination could be prevented by the addition of β-D-mannose. Likewise, the per cent phagocytosis of E. ictaluri isolate 1389 (the most intense neutrophil agglutinator) could be dramatically reduced by the inclusion of mannose in the assay mixture. Anterior kidney leucocytes and hepatocytes primarily reacted with lectins derived from Canavalia ensiformis, Ricinus communis and Triticum vulgaris. The presence on channel catfish cells of carbohydrate-lectin receptors similar in function to those found on mammalian cells and the reaction of E. ictaluri with specific lectins suggest that bacterial clearance and lectinophagocyotosis may occur in a similar manner in catfish to that in mammals.     

Serological comparison of selected isolates of Aeromonas salmonicida ssp. salmonicida

Abstract. Eight isolates of Acronionus salmonicida ssp. salmonicida were collected during furunculosis epizootics in North American Pacific coast states and provinces. Both virulent and avirulent forms of each isolate, confirmed by challenge and electron microscopy, were examined. Serological comparisons by cross-absorption agglutination tests revealed no serological differences between isolates. Using the double diffusion precipitin test, a single band was observed when antigen from a sonicated virulent strain was reacted with antiserum against a sonicated, virulent strain absorbed with homologous, avirulent strain. The presence of the single band was eliminated by excess sonication.     

Effect of temperature on growth,digestion and immunity of green,white and purple sea cucumbers,Apostichopus japonicus (Selenka)

Three color morphs (white, green and purple strains) of Apostichopus japonicus (Selenka) were cultured in artificial seawater, for approximately 90 days, in three temperature ranges: 27–22°C (high), 22–17°C (mid) and 17–12°C (low). All strains grew in all temperature ranges. Temperature significantly affected growth rate, digestibility, digestive enzymes and immune‐related enzymes. Highest specific growth rates were exhibited in 4‐month‐old sea cucumbers at mid and high temperatures, and in 16‐month‐old sea cucumbers at mid and low temperatures. Specific growth rates of green and purple strains were not significantly different, but were significantly higher than that of the white strain at mid temperatures. The digestibility of each strain was significantly higher at 27°C, 22°C and 17°C than at 12°C. Green‐strain digestibility was higher than that of purple and white strains at specific temperatures. Protease and amylase activities of all strains followed bell‐shaped temperature curves with maximum digestive enzyme activity at 17°C. The activities of alkaline and acid phosphatases were higher in the guts of the green strain than in the white or purple strains at the same temperature. Superoxide dismutase activity was higher in the purple strain than in white and green strains.     

Aeromonas salmonicida: determination of an antigen associated with protective immunity and evaluation of an experimental bacterin

Abstract. Protection against Aeromonas salmonicida was determined by passive immunization and with various bacterin preparations. Rabbit antiserum was prepared against a rough, virulent strain of A. salmonicida (AS-1R), the same strain boiled (AS-1R, boiled), and an avirulent, smooth strain of this same isolate (AS-1S). Cross-absorption, cross-passive protection and analysis by counter immunoelectrophoresis of various extraction methods were studied. It was shown that AS-1R cells contained an additional antigen not present in AS-1R (boiled) and AS-1S cells. Antiserum to the AS-1R antigen passively protected sockeye salmon, Oncorhynchus nerka (Walbaum), against a virulent challenge, and antisera to AS-1R (boiled) and AS-1S were not protective. The antigen was not destroyed by formalin or heat at 5°C for 60 min, but it appeared to be partially inactivated with proteolytic enzymes. The antigen was produced in casein yeast beef (CYB) broth up to 32 h but not thereafter, and low yields were obtained in tryptic soy or brain heart infusion (BHI) broth. It was extracted from cells with ethylenediamine tetraacetic acid (EDTA) and especially alkaline hydrolysis, but not with proteolytic enzymes or detergents. The detergents appeared to destroy the antigen. We concluded that the antigen was protein and is most likely the external A-protein (AP) reported for rough, virulent strains of A, salmonicida. Various methods of preparing A. salmonicida bacterins were evaluated by determining the level of protective immunity induced in intraperitoneally (i.p.) vaccinated fish. Growth of cells in CYB or BHI broth resulted in production of only rough (autoagglutinated in saline) variants of A. salmonicida. Although only rough variants were associated with protective immunity, one strain was not protective, it was avirulent by bath challenge. Bacterins prepared in CYB were more efficacious than those grown in BHI, but inactivation with formalin, iodine, or glutaraldehyde worked equally well. However, boiling the bacterin or filtering the cells from the bacterin removed its efficacy. Methods of releasing the AP were evaluated by sonification, pH-lysis, disaggregation and treatment with EDTA, and all treatments worked equally well. Also, precipitation on to aluminium or use of Freund's complete adjuvant did not significantly improve the protection. In parenterally vaccinated fish, protection was demonstrated by challenging the fish at various levels by bath, injection or cohabitation with infected fish. The best protection was demonstrated using the cohabitation challenge method. The potency and field efficacy of an A. salmonicida bacterin prepared in CYB broth and extracted with 5 mM EDTA was evaluated. Fish were vaccinated by i.p, injection and potency was determined in the laboratory by experimental challenge and in the field by natural challenge. Chinook salmon, O.ishawytschu (Walbaum), developed immunity within seven days at 10°C. The bacterin could be diluted up to 1:2000 without loss of potency. The field tests results were equivocal; however, (he prevalence of infection was lower in vaccinated fish.     

Virulence of Flavobacterium branchiophilum in experimentally infected salmonids

Abstract. The ability of selected strains of Flavobacterium branchiophilum to attach to and colonize the gills of five species of teleosts (four salmonid and one cyprinid) and cause mortality was investigated. In virulence studies with F. branchiophilum strain LAB4a, cumulative mortality was dose-dependent in rainbow trout, Oncorhynchus mykiss (Walbaum), and ranged from 0 to 75%. However, regardless of dose, the relative amount of gill-associated F. branchiophilum antigen increased 1 h after challenge. The gill-associated F. branchiophilum antigen in fish which survived (moribund fish) increased by four to six times compared to that detected on the gills 1 h after challenge. The gill-associated antigen on moribund fish was not significantly different from that on the gills of dead fish. Flavobacterium branchiophilum strain LAB4a also attached to the gills of brook trout, Salvelinus fontinalis (Mitchell), rainbow trout, chinook salmon, Oncorhynchus tshawytscha (Walbaum), Arctic charr, Salvelinus alpinus (L.), and common shiners, Luxilus cornutus (Mitchell), following a 1-h bath exposure, and caused mortality in all species. The virulence of eight strains of F. branchiophilum for rainbow trout was examined. Both virulent and avirulent strains adhered to the gills following bath exposure (fimbriae were observed on all strains), but only virulent strains had the capacity to further colonize the gills and cause mortality.