The Formation, Persistence, and Resistance to Disinfectant of the Erysipelothrix piscisicarius Biofilm.

Erysipelothrix piscisicarius is an emergent pathogen in fish aquaculture, particularly in the ornamental fish trade. Very little is known on the biology of this pathogen; however, the recurrence of infection and disease outbreaks after removing the fish from a system and disinfecting the tank suggest its environmental persistence. Moreover, biofilm lifestyle in E. piscisicarius has been suspected but not previously shown. The purpose of this study was to investigate the formation of biofilms on an abiotic surface in Erysipelothrix spp. We used hydroxyapatite-coated plastic pegs to demonstrate the attachment, growth, and persistence of E. piscisicarius on abiotic surfaces in both fresh and marine environments and to investigate the susceptibility of this pathogen to different disinfectants that are used in the aquaculture industry. E. piscisicarius formed biofilms that persisted significantly longer than planktonic cells did in both freshwater and saltwater over a period of 120 h (P = 0.004). The biofilms were also more resistant to disinfectants than the planktonic cells were. Hydrogen peroxide was the most effective disinfectant against E. piscisicarius, and it eradicated the biofilms and planktonic cells at the recommended concentrations. In contrast, Virkon and bleach were able to eradicate only the planktonic cells. This information should be taken into consideration when developing biosecurity protocols in aquaculture systems, aquariums, and private collections.

Improving ante mortem diagnosis of Erysipelothrix rhusiopathiae infection by use of oral fluids for bacterial, nucleic acid, and antibody detection.

Swine erysipelas is an economically important disease caused by Erysipelothrix rhusiopathiae. Pen-based collection of oral fluids has recently been utilized for monitoring infection dynamics in swine operations. The diagnostic performance of bacterial isolation, real-time PCR, and antibody detection by enzyme-linked immunosorbent assay (ELISA) and fluorescent microbead-based immunoassay (FMIA) methods were evaluated on pen-based oral fluid samples from pigs experimentally infected with E. rhusiopathiae (n=112) and from negative controls (n=32). While real-time PCR was a sensitive method with an overall detection rate of 100% (7/7 pens) one day post inoculation (dpi), E. rhusiopathiae was successfully isolated in only 28.6% (2/7 pens). Anti-Erysipelothrix IgM and IgG antibodies in pen-based oral fluids were detected at 4 to 5 dpi by FMIA and at 5 and 8 dpi by ELISA. The number of infected animals per pen, and in particular the timing of antimicrobial treatment administration impacted bacterial isolation and ELISA results. In oral fluid field samples, E. rhusiopathiae DNA was found in 23.3% of the samples while anti-E. rhusiopathiae IgG and IgM antibodies were found in 59.6% and 5.5% of the samples, respectively. The results suggest that an algorithm integrating oral fluids as specimen and real-time PCR and FMIA as detection methods is effective for earlier detection of an erysipelas outbreak thereby allowing for a more effective treatment outcome.

Comparison of conventional direct and enrichment culture methods for Erysipelothrix spp. from experimentally and naturally infected swine.

The objective of the current study was to compare the diagnostic performance of a direct isolation method for Erysipelothrix spp. with a broth-based enrichment technique. Samples were obtained from three sources: 1) experimentally inoculated pigs, 2) porcine tissue samples submitted to the Iowa State University Veterinary Diagnostic Laboratory (Ames, IA), and 3) tissues from condemned carcasses at an abattoir. Culture plates from direct isolation and broth-based technique were evaluated for growth at 24 and 48 hr. Results indicated that the broth enrichment method was markedly more sensitive for the isolation of Erysipelothrix spp. To the authors' knowledge, this is the first comparison of direct culture and broth-based enrichment methods for the isolation of Erysipelothrix spp. Interestingly, in several samples, a Gram-positive bacterium with almost identical growth characteristics to Erysipelothrix spp. was detected and identified as a Vagococcus sp. through 16S ribosomal RNA gene sequencing. The results of this study indicate that the broth-based enrichment method should be used for the isolation of Erysipelothrix spp. from clinical samples with a history suggestive of erysipelas and that Vagococcus spp. is potentially an important differential diagnosis.

Bioreactor aeration conditions modulate growth and antigen expression during Erysipelothrix rhusiopathiae cultivation.

Erysipelothrix rhusiopathiae, the causative agent of swine erysipelas, was cultivated in a 5-L stirred and aerated bioreactor under different dissolved oxygen tensions (0%, 5%, and 30% of saturation) for evaluation of the influence of oxygen on cell growth as well as on the production of the main antigenic component of the vaccine against erysipelas, a 64-69 kDa protein (SpaA). The microorganism presented different growth profiles for different aeration conditions. However, at the end of the batch cultivations, similar cell concentrations were obtained under the studied conditions. In order to maximize biomass titers and antigen production, the microorganism was cultivated in fed-batch operation mode under aerobic conditions. Under this condition, there was a fivefold increase in biomass production in comparison to the results attained in batch cultivations. To follow up antigen expression, samples collected during batch cultivations were concentrated and treated with choline for antigen extraction. Antigen expression was then assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by murine immunization tests. It was observed a direct influence of oxygen availability upon antigen expression, which is favored in the presence of oxygen. Analysis of the samples collected throughout the fed-batch process also revealed that antigen production is growth associated.

Erysipelothrix septicemia in a little blue penguin (Eudyptula minor).

On June 25, 2002, aquarium veterinarians treated a 5-year-old, male little blue penguin (Eudyptula minor) that was acutely recumbent and dull, with inappetence of 24-hour duration. The penguin died within 10 minutes of presentation despite emergency resuscitation efforts. Gross pathologic findings consisted of pulmonary congestion and intestinal hemorrhage. Histopathologic findings included necrosis of tips of intestinal villi, increased numbers of mononuclear cells in pulmonary interstitium and hepatic sinusoids, and gram-positive bacteria in systemic microvasculature. Transmission electron microscopic examination revealed short gram-positive bacilli located in lumina of glomerular capillaries and in cytoplasm of mononuclear phagocytic cells in the lung and liver. Erysipelothrix rhusiopathiae was recovered from the lung, liver, and intestine by bacteriologic culture. Amplicons from polymerase chain reaction (PCR) tests using Erysipelothrix genus-specific primers and total genomic DNA extracted from formalin-fixed, paraffin-embedded tissue sections of lung and intestine demonstrated 99% nucleotide sequence identity with 16S small-subunit ribosomal DNA of E. rhusiopathiae and E. tonsillarum. The source of infection was speculated to be fish in the diet; however, repeated attempts to detect Erysipelothrix spp. from the mucous layer of food fish using bacteriologic culture and PCR were unsuccessful. This is the first report of erysipelas in a captive aquatic bird. Details of the isolation of E. rhusiopathiae and the application of molecular testing to identify Erysipelothrix DNA in formalin-fixed, paraffin-embedded tissue sections are given.

Susceptibility of Erysipelothrix rhusiopathiae to antimicrobial agents and home disinfectants.

AIM: Erysipelothrix rhusiopathiae causes the occupationally-related infection erysipeloid in humans, and may be responsible for infections in lobster fishermen in Western Australia. There are little recent data pertaining to antimicrobial susceptibility, or susceptibility to disinfectants that might be used in the environment. The aim of this study was to determine the susceptibility of E. rhusiopathiae from human, animal and environmental sources to various antimicrobial agents and disinfectants. METHODS: The susceptibility of 60 E rhusiopathiae isolates was determined using a recommended agar dilution procedure. Susceptibility to disinfectants was achieved using a broth microdilution method. RESULTS: Penicillin and ceftriaxone, with low minimum inhibitory concentrations (MICs) (MIC90 0.03 mg/l and 0.125 mg/l, respectively), remained active against E. rhusiopathiae and should continue to be recommended for treatment. Ciprofloxacin MICs were particularly low (MIC90 0.06 mg/l), offering an alternative agent for the penicillin allergic patient. Erysipelothrix rhusiopathiae is still resistant to vancomycin (MIC90 64 mg/l), highlighting the importance of early diagnosis of E. rhusiopathiae infection in cases of endocarditis. In addition, 31 E. rhusiopathiae isolates were tested against several commercially available home disinfectants. Most were effective in killing E. rhusiopathiae with minimum bactericidal concentrations of 0.001% for Pine O Cleen, and 0.03% for Domestos, Linely and the Wheelie Bin Phenyl Cleanser. CONCLUSIONS: There appeared to be no new emergence of antibiotic resistance in E. rhusiopathiae. Various disinfectants could be used following mechanical cleaning of work environments, such as fishing boats, and equipment, to reduce the risk of infection with E. rhusiopathiae.

Preparation and partial characterization of monoclonal antibodies against the protective protein antigen of Erysipelothrix rhusiopathiae.

Thirteen mouse monoclonal antibodies (Mabs) against the protective protein antigen (P64) of Erysipelothrix rhusiopathiae were prepared and partially characterized. The titres of the Mabs varied from 200 to 1,638,400 as determined by enzyme-linked immunosorbent assay (ELISA). Of the 13 Mabs 10, two and one belonged to the IgG2a, IgG1 and IgM subclasses, respectively. All Mabs reacted strongly with the 64 kDa protein and weakly with the 43 kDa protein upon Western blotting of the alkaline extract (AE) of E. rhusiopathiae. The protective activity (PD50/ml) of the 13 Mabs against E. rhusiopathiae infection in mice varied from < 50 to > 50,000. These Mabs were classified into three groups, highly protective Mabs, moderately protective Mabs and Mabs which did not possess protective activity, based on the protective index (ratio of the PD50/ml to the antibody titre). These results suggest that the 64 kDa protein is an effective protective antigen, which is easily cleaved into many small proteins, including the 43 kDa protein, and possesses at least two epitopes related to its protective activity and at least one epitope which is not related to protection of mice against E. rhusiopathiae infection.

Isolation and purification of a protective protein antigen of Erysipelothrix rhusiopathiae.

The rapid growth and high survival rate of Erysipelothrix rhusiopathiae was determined using a culture of the bacterium in tryptic soy broth supplemented with 0.3% Tris-hydroxymethyl aminomethane and 0.1% Tween 80 (TT-TS broth). High concentrations of 64, 66 and 43 kDa proteins, which are associated with protection against E. rhusiopathiae infection in mice, were obtained by alkaline treatment of whole cells using 0.05-1 N NaOH. The supernatant of alkaline treated cells (alkaline extract; AE) was stable at alkaline or neutral pH. However, aggregates appeared at neutral pH in the absence of sodium dodecyl sulphate (SDS). A high yield of 64, 66 and 43 kDa proteins was obtained from strain Agata (serovar 5). The proteins were eluted from gel bands following SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the AE from strain Agata and designated P64 and P43. The amounts of P64 and P43 isolated were 0.7 and 0.3 mg/16 g of wet bacteria, respectively. In a mouse protection test, 50% protective doses (PD50) of P64 and P43 were 0.58 and 0.63 microgram, respectively. Upon Western blotting of the AE, both anti-P64 and anti-P43 antibodies reacted with the 64 and 43 kDa proteins. From these results, it is suggested that P64 is the most effective protective antigen and that P43 (43 kDa protein) is a degradation product of P64. Therefore, the 64 kDa structural proteins are associated with the induction of a protective activity against E. rhusiopathiae infection in mice.

Superoxide release in human fibroblasts upon treatment with culture supernatants of the arthritogenic bacteria Erysipelothrix rhusiopathiae and Mycoplasma arthritidis.

Culture supernatants of the arthritogenic bacteria Mycoplasma pneumonia, Mycoplasma arthritidis, Borrelia burgdorferi and Erysipelothrix rhusiopathiae stimulated primary cultures of human fibroblasts to release reactive oxygen species into the environment, whereas cell walls and membranes of these bacteria had no effects. Lipopolysaccharides of various gram-negative bacteria and lipid A, the lipid moiety of endotoxines, also failed to stimulate the release of reactive oxygen species by fibroblasts. The stimulatory fractions of the culture supernatants of Mycoplasma arthritidis and Erysipelothrix rhusiopathiae exhibited a molecular weight of about 9.5 kDa. After an induction period of 5 min the presence of the stimulant was not necessary any more. The primary radical released by the fibroblasts was the superoxide anion O2-. Radical formation took place continuously over some hours. Additionally, low-level chemiluminescence of fibroblasts was increased upon stimulation with culture supernatants of Mycoplasma arthritidis and Erysipelothrix rhusiopathiae. No irreversible injury of the fibroblast was caused upon stimulation and the cells exhibited normal proliferation pattern after replacing them to the culture medium.

Intracellular survival and replication of Erysipelothrix rhusiopathiae within murine macrophages: failure of induction of the oxidative burst of macrophages.

We investigated the ability of a virulent wild-type parent strain and acapsular avirulent transposon mutants to enter and survive intracellularly within murine peritoneal macrophages. In the presence of normal or immune serum, the parent and mutant strains were both ingested; however, the number of ingested bacteria was three- to fourfold greater in the case of mutant strains than in the case of the parent strain. The parent strain, but not the mutant strains, survived and replicated intracellularly when ingested in the presence of normal serum, whereas both the parent and the mutant strains were readily killed when ingested in the presence of immune serum. To further investigate the mechanism by which the parent strain can survive and replicate within macrophages, we studied the oxidative burst response of macrophages to these strains by measuring chemiluminescence and intracellular reduction of Nitro Blue Tetrazolium dye. Challenge exposure of macrophages with either the parent strain preopsonized with immune serum or the mutant strains preopsonized with normal or immune serum induced a strong oxidative burst, whereas the level was very low when the parent strain was preopsonized with normal serum. Phagocytosis of either the parent strain, in the presence of immune serum, or the mutant strains, in the presence of normal or immune serum, by macrophages reduced large amounts of intracellular Nitro Blue Tetrazolium, whereas minimal amounts were reduced by the parent strain in the presence of normal serum. These results suggest that virulent E. rhusiopathiae can survive and subsequently replicate within murine macrophages when ingested in the presence of normal serum and that the reduced production of reactive oxidative metabolites by macrophages may, in part, be responsible for this occurrence.

Growth ability and immunological properties of Erysipelothrix rhusiopathiae serotype 2.

Five field strains of Erysipelothrix rhusiopathiae belonging to serotype 2 were compared for their growth ability, immunogenicity in mice, SDS-PAGE profile of cell surface proteins and their immunoblotting patterns. Strain Tama-96 showed the most stable growth in Feist medium and tryptose phosphate broth with Tween 80 (TPB), and its immunogenicity was highest in a mouse protection test using the inactivated vaccines prepared from 20-h TPB culture. The 50% mouse protective dose of the vaccine was only 12 microliter. SDS-PAGE and immunoblotting patterns of the proteins were similar among the strains in general and indicated that 66 to 64 kDa protein antigens were dominant.

Comparison of etiological and immunological characteristics of two attenuated Erysipelothrix rhusiopathiae strains of serotypes 1a and 2.

Two acriflavine-fast attenuated Erysipelothrix rhusiopathiae strains Koganei 65-0.15 of serotype 1a (strain Kg-1a) and 2 (strain Kg-2) were comparatively characterized. Biochemical characterization showed the similar reactions with slight variation between the strains. Strain Kg-2 was more resistant to acriflavine dye than strain Kg-1a. Pathogenicity of strain Kg-2 was higher than strain Kg-1a in mice of strains ddY. C3H/He and A/J. Significant differences of clinical signs between strains Kg-1a and Kg-2 were observed in occurrence of arthritis (P < 0.05) and systemic signs (P < 0.01) of only ddY mice. C3H/He mice was more resistant than ddY and A/J mice to the infection of strains Kg-1a and Kg-2. Three culture fractions, whole culture: WC, culture filtrate: CF and killed cells: KC, of strain Kg-2 were more protective than those of strain Kg-1a in ddY mice. CF of strain Kg-2 was most protective in all fractions. Heating at 56 degrees C and 100 degrees C or treatment with trypsin completely reduced the protective activity of WC of the two strains, indicating that major protective antigens of WC were protein. The present results demonstrated that immunogenicity and pathogenicity for mice were different between the two attenuated strains.

[The development of a technology for the production of live dried vaccines against avian pasteurellosis and swine erysipelas]. / Razrabotka tekhnologii proizvodstva zhivykh sukhikh vaktsin protiv pasterelleza ptits i rozhi svinei.

The technology of the production of dried live vaccine against Pasteurella infection of fowl from Pasteur's 2nd avirulent strain, strains AB and K, has been developed. This technology includes the process of batch cultivation of Pasteurella cells, controlled in such parameters as eH, pO2 and glucose concentration, in fermenters in optimized culture medium, based on Hottinger hydrolysate and fermentative casein-yeast hydrolysate, and preservation in improved saccharose-gelatin medium prepared in potassium sulfate buffer solution. The new technology makes it possible to increase the yield of preparations with stable biological activity 5- to 13-fold in comparison with the traditional technology. Furthermore, the technology of the production of live dried vaccine against swine erysipelas from Erysipelothrix insidiosa strain BP-2 has been developed. This technology is based on maintaining the optimum conditions of the batch cultivation of E. insidiosa in meat medium based on Hottinger hydrolysate and media obtained from hydrolysate of pancreatic fermentation products of microbial biomass; the preparation thus obtained is stabilized in peptone-saccharose-gelatin medium prepared in potassium phosphate buffer solution. This increases the yield of the vaccine 8-fold in comparison with the traditional technology, while ensuring the stability of bacteria after drying and during prolonged storage.

Erysipelothrix rhusiopathiae: an occupational pathogen.

Erysipelothrix rhusiopathiae is a nonsporulating, gram-positive, rod-shaped bacterium which was identified more than 100 years ago as the etiologic agent of swine erysipelas. Since then, it has been found to cause infection in several dozen species of mammals and other animals. Humans become infected through exposure to infected or contaminated animals or animal products. By far the most common type of human infection is a localized, self-limited cutaneous lesion, erysipeloid. Diffuse cutaneous and systemic infections occur rarely. Approximately 50 cases of endocarditis have been reported; all but one recent case have involved native valves. The organism may be isolated from biopsy or blood specimens on standard culture media. It is identified by morphology, lack of motility, and biochemical characteristics; identification may be confirmed by the mouse protection test. It is susceptible to penicillins, cephalosporins, erythromycin, and clindamycin, but it is often resistant to many other antibiotics, including vancomycin, a drug frequently used in empiric therapy for infections due to gram-positive bacteria.

[The pathogenesis of erysipelas arthritis]. / Die Pathogenese der Rotlaufarthritis.

Experimental chronic erysipelas polyarthritis in rat, induced by living erysipelas bacteria, histologically can be divided into four different phases. In the phase of population bacteria are distributed diffusely within the whole joint but accumulate in the transitional zones and entheses by multiplication within the ground substance of cartilage. In the phase of acute destruction a severe inflammation of all joint tissues predominates. Bacterial antigen is eliminated by a pannus tissue destroying the cartilaginous structures. In the following phase a diffuse dystrophy of articular cartilage dominates. The reason for this process is not clear; within the cartilage bacterial antigen can seldom be demonstrated, but it accumulates intracellularly in the periphery of the joints (e.g. dense connective tissue, muscles). In the chronic phase we find a lymphoplasmacytic infiltration of the subsynovium, a lining cell hyperplasia, and pannus formation arising from the epiphyseal bone marrow cavity. The relation between chronic inflammation and destruction in the central and antigen persistence in the outer parts of the joints is a matter of current investigation.

Effect of cyclophosphamide and carrageenan on resistance of mice to Erysipelothrix rhusiopathiae.

The immunosuppressive effect of cyclophosphamide (CY) or carrageenan (CG) treatment was investigated to clarify the mechanism of resistance of mice to Erysipelothrix rhusiopathiae infection. In mice inoculated with attenuated E. rhusiopathiae, death occurred and bacterial growth in the spleen was enhanced only by CY treatment; in CG-treated mice, no death occurred and bacterial growth in the spleen was kept at a low level for at least 23 days, similar to that of nontreated control mice. These results indicated that polymorphonuclear leucocytes rather than macrophages may play an important role in the resistance of mice to E. rhusiopathiae infection.

Application of the indirect enzyme immunoassay for the detection of antibodies against Erysipelothrix rhusiopathiae.

Sera from swine and rats experimentally infected with Erysipelothrix rhusiopathiae and field sera from swine were investigated for antibodies against E. rhusiopathiae using the microtiter enzyme immunoassay (EIA) and, for comparison, the growth test (GT) and the agglutination test (AT). In principle there was a good correspondence between the results of EIA and those of the two other methods, but EIA and GT were more sensitive than AT. On the basis of the evaluation pattern of GT and AT on swine sera, EIA titers of 1/320 were considered as "chronic erysipelas titers". Compared with GT and AT, the EIA has some advantages: it is not influenced by contamination of the test sera, it takes only a few hours and using the microtiter system it is easy and economical to perform.

Comparative studies on different methods in typing strains of erysipelothrix rhusiopathiae. I. Methods and influence of some factors on their results.

To contribute to the standardization of typing Erysipelothrix rhusiopathiae strains the following points have been examined and compared: the usefulness of tube precipitation, paper chromatography and agar gel diffusion; different methods to produce good antisera (specific high titres) and antigens (CH3COOH-, HCl-extract, extraction by means of autoclavation). Rabbits have a considerably varying individual ability to produce Erysipelas antibodies with good precipitation titres. The antigenic effect of different E.rh.-strains and of dissociated forms is not equal. The density of bacteria suspension for extract production and for rabbit immunization has to be sufficient. The period of usability of extracts and antisera is different.