Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection

Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.     

Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation

The effect of growth temperature on the loss of virulence of the fish pathogen Aeromonas salmonicida was investigated. Three virulent strains were grown in Trypticase soy broth at temperatures ranging from 22 to 30 degrees C. Growth at a higher-than-optimal temperature (26 to 27 degrees C for the three strains studied) resulted in the selection of spontaneous attenuated derivatives in the initial bacterial population. For example, virulent bacteria represented less than 10% of the population of a culture grown at 30 degrees C, and attenuated derivatives were easily isolated by streaking the culture on solid medium and picking single colonies. Virulent strains autoaggregated during growth and possessed a cell wall layer (A-layer) external to the outer membrane, as previously described. Attenuated strains did not autoaggregate and did not possess the A-layer. The A-layer apparently shielded bacteriophage receptors and a mannose-specific yeast agglutinin located in the outer membrane. Thus, virulent strains exhibited impaired adsorption of phages, whereas attenuated strains were phage sensitive. Furthermore, attenuated strains agglutinated yeast cells but virulent strains did not. The attenuated strains had higher maximum growth temperatures than their virulent parent strains, and this accounts for their selection at high temperatures. It is proposed that the A-layer contributes significantly to the physical properties of the A. salmonicida cell envelope and that these physical properties of the A. salmonicida cell envelope and that these physical change upon loss of the A-layer to permit growth at a higher-than-usual temperature.     

Extracellular protectants produced by Clostridium perfringens cells at elevated temperatures

Aim:  The mechanisms of adaptation of Clostridium perfringens to high temperatures are not well understood. In this work, the involvement of extracellular compounds in protection to heat was determined.
Methods and Results:  Cells were grown in fluid thioglycollate medium or chicken broth. When mid-log phase was reached, they were heat-shocked at 50°C for 30 min. Then cultures were centrifuged and supernatants were transferred to nonshocked cells. Heat tolerance of these cells was performed at 55°C. Viable cells were determined. In some cases, supernatants were heated at 65°C or 100°C or treated with trypsin. Supernatants were fractionated and PAGE was made of fractions showing heat-protective activity. When C. perfringens was exposed to a heat shock at 50°C, extracellular factors were found in the culture supernatant that provided protection to cells not exposed to a heat shock. The extracellular factors were sensitive to heat and trypsin treatment suggesting a protein component. SDS-PAGE analysis of supernatant fractions from heat-treated cells revealed two induced proteins (56 and 125 kDa) that could be involved in heat tolerance.
Conclusion:  In this work, the presence and thermoprotective activity of extracellular factors produced by C. perfringens under a heat shock was demonstrated.
Significance and Impact of the Study:  The detection of thermoprotective extracellular factors of C . perfringens will aid in our understanding of the physiology of survival of C. perfringens in foods.     

Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis

Suicide plasmid pJB4JI, containing transposon Tn5 and phage Mu, was introduced into Aeromonas salmonicida 449 which produces a surface protein array known as the A-layer. Kanamycin-resistant exconjugants of 449 with altered ability to produce the A-layer were selected by virtue of their altered colonial morphology and color on medium containing the dye Congo red. Analysis of culture supernatants, periplasmic shock fluid, outer membranes, and whole-cell lysates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody to A-protein revealed five classes of single-insertion mutations that affected the ability of cells to produce and export A-protein and to assemble the A-layer. These studies suggest that A-protein is produced from a single chromosomal gene. The subunits subsequently pass through the periplasm and across the outer membrane. At least one gene product is required for this export. Assembly of A-layer on the cell surface then requires the presence of O polysaccharide chains on the lipopolysaccharide. In one case, insertion of Tn5 resulted in loss of ability to produce both A-protein and lipopolysaccharide with O polysaccharide chains, suggesting that synthesis of A-protein and synthesis of lipopolysaccharide may involve coordinate regulation.     

Electron microscopic observations of the phagocytosis and subsequent fate of Aeromonas salmonicida by Atlantic salmon neutrophils in vitro

Neutrophils isolated from the blood of Atlantic salmon (Salmo salar L.) were studied by electron microscopy at various time intervals after being incubated with opsonised and non-opsonised Aeromonas salmonicida strains, namely, the avirulent MT004 (A-layer negative) and the virulent MT423 (A-layer positive). After 15 min incubation with all four groups of bacteria (virulent, avirulent, opsonised or non-opsonised) a large number of neutrophils showed an elongated shape with the nucleus and all the organelles being located in one pole of the cell. Small vacuoles and clumping of glycogen granules were also observed. Neutrophils devoid of granules were noted after 30 min incubation, the majority containing engulfed bacteria. Degenerate neutrophils were also found in all the groups incubated with bacteria. Phagocytosis of bacteria was observed after 15 min incubation. The number of intracellular bacteria was very low, usually one or two per cell, although some neutrophils incubated with the opsonised avirulent strain MT004 contained a larger number of engulfed bacteria. Ingestion of bacteria was usually accompanied by the formation of phagocytic vacuoles containing an amorphous material of moderate electron-density as well as granule discharge into the vacuole. Both strains (MT004 and MT423), opsonised and non-opsonised, underwent morphological alterations after 3-7 h incubation suggesting that both A. salmonicida strains were killed by the neutrophils.     

Role of adherent cells in dextran sulfate-mediated enhancement of mitogen-induced B-cell proliferation

We have studied lipopolysaccharide (LPS)-induced proliferation in the rat and have found that the addition of the compound Dextran sulfate (DxS), which itself is not mitogenic, to LPS stimulated cultures results in significant enhancement of cell division. A "DxS-free" supernatant from DxS stimulated spleen cell cultures is able to substitute for DxS in stimulatory activity. This supernatant possesses interleukin 1 (IL-1) activity, however, the addition of purified recombinant IL-1 to LPS-stimulated cultures does not result in augmentation of proliferation. A DxS-free supernatant from DxS stimulated adherent cells is also able to substitute for DxS in stimulatory activity. The active molecule(s) present in the adherent cell-derived DxS-free culture supernatant appears to be distinct from classical IL-1.     

Modulation of the activity of sea bass (Dicentrarchus labrax) head-kidney macrophages by macrophage activating factor(s) and lipopolysaccharide

The aim of this study was to establish the requirements for macrophage activating factor (MAF) production by sea bass head-kidney leucocytes and the kinetics of macrophage activation when exposed to MAF-containing supernatants and/or lipopolysaccharide (LPS), a known macrophage stimulant. MAF activity was found in culture supernatants of total head-kidney leucocytes pulsed with 5 microg ml(-1)Con A, 5 or 10 ng ml(-1)PMA and 100 ng ml(-1)calcium ionophore, or 10 microg ml(-1)Con A alone, as assessed by the capacity to prime macrophages for enhanced production of reactive oxygen intermediates (ROI). Mixed leucocyte cultures from two or eight fish showed higher MAF activity after stimulation, indicating that a mixed leucocyte reaction was also important for MAF production. MAF-induced activation of macrophage cultures was highest at 18 h of exposure and was lost by 72 h except for MAF induced by Con A-stimulation alone. LPS primed macrophages for increased ROI production at early incubation times and down-regulated ROI production after 24 h. LPS had no effect in further stimulating the MAF-induced priming effect on production of ROI and down-regulated the MAF-priming by 48 h. Sea bass head-kidney macrophages did not show increased nitrite production when exposed to MAF and/or LPS, which may be related to their differentiation status.     

Comparison of the surface properties of seven strains of a fish pathogen, Aeromonas salmonicida

Several physical properties related to the surface characteristics of autoaggregating and non-autoaggregating strains of Aeromonas salmonicida have been investigated. Properties examined included resistance to the bactericidal action of trout serum, adhesion to fish leucocytes and fish cell monolayers in vitro , resistance to phagocytosis by fish leucocytes and the in vivo localization following intraperitoneal injection. For each strain the presence or absence of an extracellular protein A-layer was investigated and the pathogenicity for brook trout determined.
Presumptive A-layer protein, in the form of a 49 kdal subunit, could be detected only in one of the strains examined. This strain autoaggregated and was the most resistant to serum bactericidal activity. Complement activation by the alternative pathway was thought to be responsible for this heat-labile bactericidal activity. Three strains that autoaggregated and three that did not had no detectable A-layer. Autoaggregating strains appeared more adhesive to both fish cell types but all strains were phagocytosed by fish leucocytes to a similar degree. An autoaggregating strain was localized in the spleen. The seven strains were only moderately pathogenic for brook trout, possibly as a result of the challenge system. In view of this, no property investigated could be correlated with greatly increased virulence.     

Lipopolysaccharide changes in smooth-type avirulent Shigella in comparison with initial virulent strains]

The comparative study of the lipopolysaccharides (LPS) of virulent and avirulent strains of S. sonnei, phase I (smooth colonies), has been made. Electrophoresis of LPS and subsequent densitometry of electrophoregrams have revealed the increase of the fraction of long 0-chains with a considerable number of recurring elements in 2 out of 3 LPS preparations obtained from avirulent shigellae. In mice immunized with these LPS preparations a considerably greater number of antibody-producing cells can be detected in Jerne's test on sheep red blood cells (SRBC) sensitized with the LPS of a virulent strain than on those sensitized with the above LPS preparations. Long 0-specific chains supposedly inhibit the fixation of individual complement components on the corresponding sensitized SRBC. The LPS of the third avirulent strain of S. sonnei, phase I, with transposon integrated into its genome, which has led to the formation of the avirulent variant of a previously virulent strain, seems to contain fine structural differences from the initial virulent strain. The immunogenicity of the LPS of this avirulent strain is greatly (3-4 times) decreased, which is manifested by the number of antibody-producing cells detected in Jerne's test on SRBC sensitized with LPS preparations obtained from these two strains.     

Screening microorganisms for insulin binding reveals binding by Burkholderia multivorans and Burkholderia cenocepacia and novel attachment of insulin to Aeromonas salmonicida via the A-layer

Exposure to microorganisms is considered an environmental factor that can contribute to Type 1 diabetes. Insulin-binding proteins (IBPs) on microorganisms may induce production of antibodies that can react with the human insulin receptor (HIR) with possible consequences in developing a diabetic autoimmune response against HIR and insulin. The interaction of insulin with microorganisms was studied by screening 45 microbial species for their ability to bind insulin. Binding assays were performed using labelled insulin to identify insulin-binding components on the microorganisms. Burkholderia multivorans and Burkholderia cenocepacia isolated from patients with cystic fibrosis (CF) and the fish pathogen Aeromonas salmonicida were the only strains of those tested, which showed insulin-binding components on their cell surfaces. Further work with A.?salmonicida suggested that the insulin-binding activity of A.?salmonicida is due to the A-layer. A mutant of A.?salmonicida lacking the A-layer showed binding, but at a much reduced rate suggesting another insulin-binding component in addition to the high affinity of the A-protein. Soluble protein lysates were subjected to Western ligand blotting using peroxidase-labelled insulin to detect IBPs. Two positive IBPs were apparent at approximately 30 and 20?kDa in lysates from Burkholderia strains, but no IBP was detected in A.?salmonicida lysates.     

Lipopolysaccharides of Shigella sonnei

Immunobiological properties of native lipopolysaccharides (LPS) from virulent and avirulent strains of Shigella sonnei bacteria (LPS-V and LPS-A, respectively) were studied. In avirulent bacteria, LPS-V induced immunosuppressive activity specific of the virulent strain. LPS of the avirulent strain, whereas LPS-A lacked this property. Native LPS-V with immunosuppressive activity were isolated from the virulent strain by and immune affinity method. Treatment of LPS-V with phenol or TCA abolished its activity and converted it into the LPS-A form. The data showed that LPS-A can be converted back to the LPS-V form by redox treatment. This approach seems to be promising for activating LPS extracted from cells with TCA or a water-phenol mixture.     

Porphyrin binding by the surface array virulence protein of Aeromonas salmonicida.

Congo red binding by virulent A-layer-containing (A+) and avirulent A-layer-deficient (A-) strains of Aeromonas salmonicida was examined. Congo red binding to A+ cells was enhanced by salt and thus hydrophobically driven, but at low Congo red concentrations binding was salt independent. Congo red was bound by A+ cells by a kinetically distinct mechanism (Kd, 0.25 microM) which was absent in A- isogenic strains. Purified A-layer protein ("A protein") protein A also bound Congo red with similar affinity (Kd, 0.40 microM). Congo red binding was structurally specific; it was not influenced by a wide variety of compounds including amino acids and nucleotides and only weakly inhibited by structurally similar dyes. However, protoporphyrin IX and hemin were strong competitive inhibitors of Congo red binding. Protoporphyrin and hemin were bound only by A+ strains (KdS of 0.41 and 0.63 microM, respectively). Furthermore, binding of these porphyrins was strongly inhibited by Congo red but weakly inhibited by hematoporphyrin. Purified A protein also bound protoporphyrin IX and hemin with affinities similar to those of A+ cells (KdS of 0.94 and 0.41 microM, respectively.     

Suppression of lymphocyte reactivity by culture supernatant from horse embryos and endometrium

The mechanisms that permit maternal tolerance of the conceptus allograft during early pregnancy in the mare have not been investigated. Embryos and endometria were collected from mares 14 days after ovulation and cultured for 20.5 h. The effect of addition of culture supernatant on incorporation of [3H]thymidine by equine peripheral blood lymphocytes was studied. Culture supernatant from endometrium of nonpregnant mares did not affect lymphocyte blastogenesis, but supernatant from both embryos and endometrium of pregnant mares reduced concanavalin A (Con A)- and phytohemagglutinin-induced blastogenesis. Five of six cultures performed in the present of indomethacin did not contain immunosuppressive factors. The suppressive effect on Con A-induced blastogenesis was eliminated by charcoal treatment of the supernatants and reduced by treatment with trypsin or heat. Blastogenesis of bovine lymphocytes was inhibited by culture supernatant of endometrium from pregnant mares, but not by embryo supernatant. Preincubation of blood lymphocytes with supernatants from endometrium of pregnant mares enhanced subsequent incorporation of [3H]thymidine by lymphocytes. A 24-h delay in addition of embryo culture supernatants significantly reduced the degree of immunosuppression. These results suggest that probably more than one substance interacts with the lymphocyte cultures and the observed blastogenesis reflects the end result of the interaction between suppressive and stimulating factors. The lymphocyte inhibitory effect evident in supernatants from embryos and endometrium from pregnant mares may be important in local immunosuppression and maternal acceptance of pregnancy.     

Congo red agar, a differential medium for Aeromonas salmonicida, detects the presence of the cell surface protein array involved in virulence.

Strains of the fish pathogen Aeromonas salmonicida which possess the cell surface protein array known as the A-layer (A+) involved in virulence formed deep red colonies on tryptic soy agar containing 30 micrograms of Congo red per ml. These were readily distinguished from colorless or light orange colonies of avirulent mutants lacking A-layer (A-). The utility of Congo red agar for quantifying A+ and A- cells in the routine assessment of culture virulence was demonstrated. Intact A+ cells adsorbed Congo red, whereas A- mutants did not bind Congo red unless first permeabilized with EDTA. The dye-binding component of A+ cells was shown to be the 50,000-Mr A-protein component of the surface array. Purified A-protein avidly bound Congo red at a dye-to-protein molar ratio of about 30 by a nonspecific hydrophobic mechanism enhanced by high salt concentrations. Neither A+ nor A- cells adsorbed to Congo red-Sepharose columns at low salt concentrations. On the other hand, A+ (but not A-) cells were avidly bound at high salt concentrations.     

Characteristics of 'atypical', cytochrome oxidase-negative Aeromonas salmonicida isolated from ulcerated flounders (Platichthys flesus (L.))

'Atypical', cytochrome oxidase-negative variants of the fish pathogen Aeromonas salmonicida , isolated from ulcerated flounder ( Platichthys flesus ), were studied using different methods. Two of the strains possessed a protein that corresponded to the A-layer protein of Aer. salmonicida . The strains reacted with antibodies against the A-layer and monoclonal antibodies against the O-antigen of typical Aer. salmonicida . These tests confirm that the isolates from flounder should be classified as Aer. salmonicida . Analysis of the fatty acids showed that the isolates were rather homogenous but the values of the guanine plus cytosine content of the DNA of the bacteria varied too much for any conclusion to be drawn on their taxonomic location. The strains examined exhibited several biochemical characters that differed from those of the type strains of Aer. salmonicida subsp. salmonicida and Aer. salmonicida , subsp. achromogenes . The results suggest that these 'atypical', cytochrome oxidase-negative variants may form a new subspecies of Aer. salmonicida .     

The effect of cyclodextrin on lipopolysaccharide production in cultures of Bordetella pertussis

The effect of adding 500 micrograms of (2,6-0-dimethyl) beta-cyclodextrin (Me-beta-CD) per ml of Stainer-Scholte (SS) medium in two-day shaker flask cultures of Bordetella pertussis on the production of lipopolysaccharide (LPS) was investigated. The amount of LPS per 10(9) cells found in the supernatants of these cultures was either somewhat reduced or unaffected by comparison with the amounts in cultures grown in SS-medium alone. In addition, the time course of LPS release from cultures of B. pertussis strain 3843 cells during a 96-h growth period in normal and Me-beta-CD-enriched SS medium is described. By using the enriched medium bacterial growth, the production of filamentous haemagglutinin (FHA) and of pertussis toxin (Pt) and the levels of haemagglutination and lymphocytosis-promoting activity were enhanced to various degrees. Measurements made on sedimented whole and on sonicated B. pertussis cells grown in the two media showed no differences in LPS content. The reasons for the reduced/unaffected LPS production are discussed. It has been suggested that an interaction between hydrophobic cavities of the Me-beta-CD molecules and the 'lipid A' part of LPS reduces the reactivity of LPS in the Limulus Amoebocyte Lysate (LAL) assay. This possibility, however, was rejected as the reactivity of Me-beta-CD-spiked purified B. pertussis strain 3803 LPS, compared with unspiked samples, remained unchanged.     

Analysis of human T cell responses to group A streptococci using fractionated Streptococcus pyogenes proteins

Cell extract and spent culture supernatant proteins from Streptococcus pyogenes Manfredo strain (type M5) were each separated to give 22 narrow range molecular weight fractions by blot-elution from SDS-polyacrylamide gels. Eluted samples and unfractionated proteins were screened for T cell stimulatory activity using human peripheral blood mononuclear cells (PBMC) from healthy adults in proliferation assays. Responses were measured in 4- and 7d cultures. Responses to a wide range of cell extract proteins were revealed by fractionation, the degree of response to each fraction varying between donors. Unfractionated culture supernatant proteins elicited proliferative responses by PBMC from all individuals examined. Responses to culture supernatant fractions containing 25–33 kDa proteins could be attributed to known superantigens. Furthermore, samples from culture supernatants containing higher molecular weight fractions (>45 kDa) elicited responses in 50% of donors in 7d cultures, suggesting that these fractions contained common recall antigens. The efficacy of using electroeluted samples to identify T lymphocyte stimulatory proteins was confirmed by demonstrating that a known superantigen of S. pyogenes Manfredo strain, streptococcal pyrogenic exotoxin C (SPEC), could be fractionated successfully using this method and its activity recovered. Our results show that human T cell responses to group A streptococci involve a remarkably wide range of both cell-associated and released streptococcal proteins.     

Conjugational transfer of genes determining plant virulence in Erwinia amylovora.

A stable virulent donor strain (EA 178R1-99) of Erwinia amylovora can transfer, by conjugation during a 3-h mating period, the gene or genes which determine(s) plant virulence to avirulent recipient strains (EA178-M64S1 and EA178-M173S1) of Escherichia amylovora. The virulence of over 200 recombinant clones was tested; they all were as virulent on immature Bartlett pear fruits (and, in the smaller series of strains tested, also, on Pyracantha twigs) as was the parent donor strain. Although the avirulent recipeint strains are amino acid auxotrophs, addition of the required amino acids to the inocula in plant virulence trials does not of itself restore virulence. Two small series of prototrophic revertant clones were selected from the auxotrophic avirulent recipient strains; only nine of the 21 prototrophic revertant clones regained virulence, whereas the other 12 prototrophic revertant clones remained avirulent, again suggesting a lack of parallelism between nutritional status and virulence in this system. Preliminary interrupted mating trials, carried out at 15-min intervals over 3 h, show that ser is transferred during the first 15 min, that pro starts entering at about 75 min (and with a higher frequency later), and that lac (originating from an integrated Escherichia coli F'lac) enters toward the end of the 3-h mating period and at a reduced frequency compared to the other markers. The gene or genes which determine(s) plant virulence in this Escherichia amylovora donor strain appear(s) to be transferred readily and seemingly completely to recipient strains during the first 15 min of a 3-h mating period. Exposure of the virulent donor strain to acridine orange or ethidium bromide does not result in loss of virulence, suggesting (but, of course, not proving conclusively) that the determinant(s) of virulence in Escherichia amylovora might be chromosomal rather than extrachromosomal.     

Siderophore production by Aeromonas salmonicida.

Growth under conditions of iron-restriction and the production of siderophores was examined in 21 typical and 14 atypical strains of Aeromonas salmonicida. With the exception of one atypical strain, all strains grew and multiplied in the presence of the high-affinity iron chelators ethylenediamine di(o-hydroxyphenylacetic acid), alpha, alpha'-dipyridyl or transferrin. Chrome azurol S agar was used to screen bacterial strains growing under these conditions for the production of siderophores. Siderophore production was detected only in the typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains. Siderophores were also detected in the iron-restricted culture supernatants of typical strains, where they were associated with an iron-binding activity. The siderophore was extracted from iron-restricted culture supernatant of one strain by adsorption onto an XAD-7 resin; it behaved as a 2,3-diphenol-catechol in several colorimetric assays. The results indicate that although both typical and atypical strains of A. salmonicida grow and multiply under conditions of iron-restriction, they use different iron-uptake mechanisms, siderophore-mediated and siderophore-independent, respectively. In cross-feeding assays, growth of typical strains was stimulated only by homologous iron-restricted supernatant, suggesting strain differences in the siderophore produced. However, one strain produced a culture supernatant with growth-stimulating activity for other typical and also atypical strains.     

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells

Abstract Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent β-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent β-lactam-resistant strains persisted within macrophages for at least 18 min only. A β-lactam-resistant mutant was obtained from a β-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, β-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.