I th l r e d im University, Ames, IA 50011 and †John G. Shedd Aquarium, 1200 South Lake Shore Drive, Chicago, IL 60605, USA Summary Keywords: Erysipelothrix spp.; fish; harbour seal; pig Er der rods with worldwide distribution. The main o n hi et al., 1987a). In addition, there are other strains 11, 12, 15, 16, 17, 19, 21 and N; E. tonsillarum contains J. Comp. Path. 2013, Vol. 148, 365e372 Available online at www Much work has been conducted to characterize host for Erysipelothrix spp. is the pig (Wang et al., 2010) and this bacterium continues to have a major economic impact for pig production systems world- wide. Three clinical presentations are recognized in pigs: the acute form characterized by sudden illness/ death and often by rhomboid or diamond-shaped skin lesions, the subacute form that is typically less se- vere than the acute form and sometimes subclinical, and the chronic form characterized by vegetative en- docarditis and arthritis (Grieco and Sheldon, 1970). that constitute one or more additional species cur- rently known as Erysipelothrix species 1 (Takahashi et al., 1992, 2008), Erysipelothrix species 2 (Takahashi et al., 1992, 2008), Erysipelothrix inopinata (Verbarg et al., 2004), and Erysipelothrix species 3 (Takahashi et al., 2008). Furthermore, Erysipelothrix spp. can be divided into at least 28 known serotypes. E. rhusiopathiae includes serotypes 1a, 1b, 2, 4, 5, 6, 8, 9 (a majority of strains), Er Cor 002 http Introduction ysipelothrix spp. are facultative gram-positive slen- The genus Erysipelothrix can be subdivided into tw major species: Erysipelothrix rhusiopathiae (Skerma et al., 1980) and Erysipelothrix tonsillarum (Takahas ysip resp 1-99 ://d In order to determine the diversity and pathogenicity of Erysipelothrix spp. isolates recovered from marine fish, a harbour seal (Phoca vitulina) and the marine environment, 14 isolates were characterized by genotyping, se- rotyping, determination of the surface protective antigen (spa) gene type and assessment of virulence in a pig bioassay. All 14 isolates were Erysipelothrix rhusiopathiae. Isolates were determined to be of serotypes 2 (n ¼ 3), 3 (n¼ 1), 4 (n¼ 1), 12 (n¼ 1), 15 (n¼ 1) or 21 (n¼ 6), and one isolate cross-reacted with serotypes 5 and 21. The spa gene analysis determined that 64.3% (n¼ 9) were spaAand 35.7% (n¼ 5) were spaB1. In pigs, 10/14 isolates induced small plaques to diamond-shaped cutaneous lesions consistent with Erysipelothrix spp. infection. The results of this study indicate that the marine E. rhusiopathiae isolates have greater genetic and antigenic diversity than pig isolates and are capable of inducing classical skin lesions in pigs. � 2012 Elsevier Ltd. All rights reserved. EXPERIMENTALLY Erysipelothrix rhusiopa from Fish, a Harbour Sea Marine Environment a Characteristic Cutan T.Opriessnig*, H. G. Shen*, J. S. Ben *Department of Veterinary Diagnostic and Production An elothrix spp. at the genetic and antigenic levels. ondence to: T. Opriessnig (e-mail:
[email protected]). 75/$ - see front matter x.doi.org/10.1016/j.jcpa.2012.08.004 NDUCED DISEASE iae Isolates Recovered (Phoca vitulina) and the e Capable of Inducing ous Lesions in Pigs er*, J. R. Boehm† and P. G. Halbur* al Medicine, College of Veterinary Medicine, Iowa State .sciencedirect.com www.elsevier.com/locate/jcpa serotypes 3, 7 (a majority of strains), 10 (a majority of strains), 14, 20, 22, 23, 24, 25 and 26; Erysipelothrix sp. 1 contains serotype 13; Erysipelothrix sp. 2 contains se- rotype 18 and few strains of serotypes 9 and 10; Erysi- pelothrix sp. 3 contains some strains of serotype 7; and E. inopinata has not been characterized serologically � 2012 Elsevier Ltd. All rights reserved. (Takahashi et al., 1987b, 1992, 2008; Verbarg et al., 2004). In 1998, a surface protective antigen (spa) gene was identified in Erysipelothrix spp. and found to express SpaA protein (Makino et al., 1998). In 2007, Spa pro- teins were further classified into three main types, SpaA, SpaB and SpaC (To and Nagai, 2007) and in 2010 two distinguishable clusters of spaB, spaB1 and spaB2 were identified (Shen et al., 2010). To date, the Spa antigen is one of the best characterized surface proteins of Erysipelothrix spp. and is associated with protection against clinical disease (Ingebritson et al., 2010; To et al., 2010). Besides its importance for pigs, Erysipelothrix spp. is also capable of infecting a variety of birds, fish and shellfish (Veraldi et al., 2009). In marine fish, Erysipelothrix spp. cause no recognizable disease, but they can survive for extended periods of time on the mucoid exterior slime of fish (Wood, 1975). To date, little is known about the genetic and anti- genic properties and pathogenicity of these marine Erysipelothrix spp. isolates. The aim of this study was to determine the diversity and pathogenicity of Erysipelothrix spp. isolates recovered from marine Materials and Methods Erysipelothrix spp. Isolates The 14 Erysipelothrix spp. isolates utilized in this study are summarized in Table 1. All of the isolates were re- covered between 1995 and 2004 from the surface of feed fish (capelin, small herring, large herring, fresh caught as batches of a single species, subsequently fro- zen, thawed and then used as a food source in an aquarium), from swabs of buckets containing feed fish or from swabs taken twice (10 months apart) from the gingival surface of a captive harbour seal with no clinical sign of illness. Serotyping Serotyping was done on all Erysipelothrix spp. isolates prior to inoculation and on isolates that were recov- ered from the pig skin biopsies at termination of the ex- periment. In brief, a pure culture was grown at 37�C for 36 h in 30 ml of heart infusion broth (Becton Dick- inson, Sparks, Maryland, USA) supplemented with 10% horse serum (SigmaeAldrich, St Louis, Mis- souri, USA) (Wood and Harrington Jr., 1978). The ble la nti ae ae ae 2 A Large Severe + + + ae ae ae ae ae ae ae ae ae ae ae al 366 T. Opriessnig et al. fish, a harbour seal (Phoca vitulina) and marine envi- ronmental samples by genotyping, serotyping, de- termination of the spa type and by testing the isolates in a bioassay in pigs. Ta Erysipelothrix spp. iso No. Sample ID Origin Date isolated Genetic and a Genotype 1 Capelin 1 Feed fish 10/07/00 E. rhusiopathi 2 Large herring 1 Feed fish 30/10/00 E. rhusiopathi 3 Small herring Feed fish 27/03/00 E. rhusiopathi 4 Capelin (C2TO) Feed fish 03/08/04 E. rhusiopathi 5 Large herring Feed fish 21/0700 E. rhusiopathi 6 Gingival swab (orange) Harbour seal 03/12/01 E. rhusiopathi 7 Water from bottom of feed bucket (A5-262) Feed fish 02/03/95 E. rhusiopathi 8 Herring T1-1 Feed fish 18/05/04 E. rhusiopathi 9 Capelin 2 Feed fish 21/08/00 E. rhusiopathi 10 Large herring 2 Feed fish 06/11/00 E. rhusiopathi 11 Gingival swab (orange) Harbour seal 19/02/01 E. rhusiopathi 12 Large herring 3 Feed fish 07/08/00 E. rhusiopathi 14 Capelin 2 Feed fish 12/06/00 E. rhusiopathi 14 Water from bottom of feed bucket (A2) Feed fish 01/03/01 E. rhusiopathi IHC, immunohistochemistry; -, negative; +, positive. *Size of the gross lesions was determined and categorized as normal, sm †Microscopical lesions in each pig were scored for severity of suppurative d score ranging from 0 to 7 was calculated. Dermal sections were considered moderate lesions (score ¼ 3 or 4) or severely affected (score ¼ 5, 6 or 7). 3 B1 Small Moderate e e + 4 B1 Small Mild e e e 12 B1 Small Moderate + e e 15 B1 None None e e e 21 A Large Moderate e + + 21 B1 None None e e + 21 A None None e e + 21 A Large Severe e e + 21 A Small Moderate e e + 21 A Large Moderate e e + 5, 21 A None None e e e l plaque (#25 mm) or large plaque (>25 mm). culture was then killed by adding a formalin solution with a final concentration of 1% (SigmaeAldrich), held at room temperature for 12 h, harvested by cen- trifugation and washed twice in 0.85% NaCl solution 1 tes used in this study genic characterization Pathogenicity in pigs Serotype Spa-type Gross lesions* Microscopical lesions† IHC Direct isolation Enrichment 2 A Small Mild e e + 2 A Large Severe + + + ermatitis (0e3), ulceration (0, 1) and vasculitis (0e3) and an overall normal (overall score¼ 0), mildly affected (score¼ 1 or 2), affected by containing 0.5% formalin. Washed cells were sus- pended in 1.5 ml of distilled water and autoclaved at 121�C for 1 h (Wood and Harrington Jr., 1978). The supernatant was collected and used for the agar gel precipitation test (Mansi, 1958). Homologous pos- itive controls were used with each test run. Reactions were recorded after 24 h (Wood and Harrington Jr., 1978). Genotyping The genotype origin of each isolate was determined by a multiplex polymerase chain reaction (PCR) ca- pable of detecting and differentiating amongE. rhusio- pathiae, E. tonsillarum and Erysipelothrix sp. 2 (Pal et al., 2009). Spa Typing The spa type of each isolate was determined by a mul- pathogens, including swine influenza virus, porcine arrival at the research facility, serum samples were collected and tested by an in-house enzyme-linked immunosorbent assay (courtesy Dr. J. Huchoppa) for the presence of anti-Erysipelothrix spp. antibodies, and the pigs were found to be negative. The pigs were housed in the same room in one pen. The pigs were monitored and allowed to acclimatize prior to inoculation. At the time of inoculation, the pigs were approximately 8 weeks old. Each of the selected Erysipelothrix spp. strains was prepared in brain heart infusion (BHI) broth (Becton Dickinson) supplemented with 5% fetal calf serum (FCS) (Becton Dickinson) and plated on BHI agar (Becton Dickinson) containing 5% FCS. Pretrial standard plate counts were performed to determine log phase growth within 30e60 min after harvest and to estimate bacterial concentrations that were subsequently adjusted to log710 colony-forming units/ ml. Purity of the inocula was assured by culture and Pathogenicity of Marine Erysipelothrix spp. Isolates in Pigs 367 reproductive and respiratory syndrome virus andMy- coplasma hyopneumoniae. E. rhusiopathiae vaccination was not used in the breeding stock on the source farm. On tiplex real-time PCR capable of detecting and differ- entiating among spaA, spaB1, spaB2 and spaC genes (Shen et al., 2010). Pig Bioassay The experimental protocol was approved by the Iowa State University Institutional Animal Care and Use Committee. Two 2-week-old conventional pigs were purchased from an isolated herd free of major swine Fig. 1. Location of the injection sites for each Erysipelothrix strain used identification by standard methods (Takahashi et al., 1992). Challenge was performed as described by Wood (1979). In brief, each animal was restrained; the skin section selected for inoculation was cleaned by brush- ing with mild soapy water and circled using a black marker (Fig. 1). Each Erysipelothrix spp. isolate was tested by intradermal injection of 0.1 ml into the right or left abdominal skin (Fig. 1). The distances between injection sites were at least 15 cm in any direction. The pigs were humanely euthanized by intrave- nous pentobarbital overdose (Fatal-Plus� Vortech Pharmaceutical, Dearborn, Michigan, USA) at 48 h post inoculation and subjected to complete in this study. Skin sections were evaluated by a veterinary pa- seen in the abdominal or thoracic cavity of either pig. diverse and 10 of 14 were capable of inducing cutane- sn thologist (TO) without knowledge of the inoculation status. Sections were scored for the presence and se- verity of suppurative dermatitis (0, absent to 3, severe and diffuse), presence of epidermal ulceration (0, ab- sent; 1, present) and the presence and severity of vas- culitis (0, absent to 3, severe and diffuse). The three scores for each pig were added together to derive an overall score ranging from 0 to 7. The overall dermal lesion scores were considered normal or no lesion (overall score ¼ 0), affected by mild lesions (score ¼ 1 or 2), affected by moderate lesions (score¼ 3 or 4) or severely affected (score¼ 5, 6 or 7). Immunohistochemistry (IHC) for detection of Ery- sipelothrix spp.-specific antigen was performed on formalin-fixed and paraffin wax-embedded sections of skin (Opriessnig et al., 2010) using pooled rabbit antiserum against E. rhusiopathiae serotypes 1a, 1b and 2. A positive control slide (from a clinically af- fected pig) and a negative control slide (fromahealthy pig) were included in each experiment. Scoring was done by a veterinary pathologist (TO) without knowledge of the inoculation status. Scores ranged from 0 (no signal) to 3 (abundant antigen present). Direct Erysipelothrix spp. culture was performed on homogenized sections of skin (Bender et al., 2009) on trypticase soy agar containing 5% sheep blood (Thermo Fischer Scientific Remel Products, Lenexa, Kansas, USA) and colistin-nalidixic acid agar con- taining 5% sheep blood (Thermo Fischer Scientific Remel Products). Plates were incubated aerobically at 35�C and examined at 24 and 48 h post inocula- tion. In addition to direct culture, the tissue homoge- nates were also enriched using an Erysipelothrix spp. selective broth (Bender et al., 2009). Results Characterization of Erysipelothrix spp. Strains The results obtained with the Erysipelothrix genotype- specific multiplex real-time PCR indicated that all of the isolates used in this study were E. rhusiopathiae (Table 1). The following serotypes were identified in necropsy examination. The absence or presence and degree of gross lesions (redness, vesicle formation) on the inoculation areas and plaque formation were categorized as small (#25 mm in diameter) or large (>25 mm in diameter). Sections of all inoculation sites were collected in individually labelled jars, fixed in 10% neutral buffered formalin and processed rou- tinely. 368 T. Opries the isolates: serotype 2 (n ¼ 3), serotype 3 (n ¼ 1), se- rotype 4 (n ¼ 1), serotype 12 (n ¼ 1), serotype 15 (n ¼ 1), serotype 21 (n ¼ 6) and one isolate cross re- ous lesions in pigs. The diversity of the isolates was confirmed by com- parison of genotypes, serotypes and expected spa types. The serotype 3 isolate used in this study, recov- Visible skin lesions and plaques of variable forms, characterized by local urticarial lesions ranging in size from 18 to 64 mm after 48 h (Table 1, Fig. 2), were induced by 10 of the 14 isolates. The microscop- ical lesions associated with these 10 isolates varied from mild to severe suppurative inflammation and vasculitis with or without ulceration (Fig. 3). Erysipe- lothrix spp. antigen was demonstrated within the skin lesions induced by three (serotype 2 and 12) of these 10 isolates as determined by IHC (Table 1, Fig. 3). Skin lesions characteristic of E. rhusiopathiae infec- tion were not seen with four of the 14 isolates by 48 h after inoculation. These isolates also did not in- duce microscopical lesions and E. rhusiopathiae antigen was not detected. Direct attempts to isolate viable bacteria from the skin sections resulted in isolation of E. rhusiopathiae from three of these 10 samples from sites with skin le- sions, while direct isolation from sites without visible lesions was unsuccessful. When enrichment tech- niques were utilized, the isolation success increased to eight of the 10 samples from sites with gross lesions and to two of the four sites without gross ormicroscop- ical lesions (Table 1). All isolates recovered were sero- typed and confirmed to be the same serotype that was used for inoculation of the respective sites. Discussion In this study, 14 Erysipelothrix spp. isolates of marine origin were characterized genetically, antigenically and for their pathogenicity in a pig skin injection model. The isolates from fish, a harbour seal and the marine environment were found to be genetically acted with serotypes 5 and 21 (Table 1). The spa types determined are summarized in Table 1. Of the 14 iso- lates, 9/14 were positive for spaA and 5/14 were posi- tive for spaB1. Pathogenicity of the Erysipelothrix spp. Strains Clinical disease was not observed in either of the two pigs within the 48 h observation period between inoc- ulation and necropsy examination. Gross lesions con- sistent with systemic erysipelas (enlarged spleen, petechial haemorrhages on organ surfaces) were not ig et al. ered from the surface of the capelin, was E. rhusiopa- thiae and was positive for spaB1. Previously, the reference strain for serotype 3, Wittling E1 (originally ip Pathogenicity of Marine Erys obtained fromG.Wellmann, Berlin, Germany;Wood and Harrington Jr., 1978) was determined to be E. tonsillarum and was negative for all known spa types (Shen et al., 2010). Moreover, isolates within serotype 12 and 15 were positive for spaB1 in the present study, which is in contrast to the previous pig-origin refer- ence strains of serotypes 12 and 15, which were posi- tive for spaA (Shen et al., 2010). Interestingly, another study, using the same serotype 12 isolate, fur- ther confirmed the negative spaA status of this isolate (Ingebritson et al., 2010). In addition, 5/6 serotype 21 isolates were positive for spaA while only 1/6 isolates followed the previously described spa pattern and was positive for spaB1 (Shen et al., 2010). Overall, the results obtained are in agreement with a previous study (Ingebritson et al., 2010), which also found that the spa types of marine origin are more variable than those of terrestrial origin. Presence of Erysipelothrix spp. in fish or the marine environment poses a major risk for cetaceans due to their high susceptibility. The disease presents as pera- cute to acute or subacute forms of septicaemia and has Fig. 2. Local urticarial lesions in pigs induced by selected Erysipelothrix surface of a harbour seal. (B) Isolate 8 recovered from the surfa herring. (D) Isolate 14 recovered from the surface of a capelin elothrix spp. Isolates in Pigs 369 been documented in several species of dolphins (Geraci et al., 1966; Thurman et al., 1983; Buck and Spotte, 1986; Kinsel et al., 1997). The peracute/acute form of the disease in marine mammals occurs with few or non-specific clinical signs, and most often re- sults in death before clinical signs occur (Medway, 1980; Lacave et al., 2001). Captive cetaceans in par- ticular are prone to develop peracute or acute septi- caemia associated with Erysipelothrix spp., which is thought to be due to access to contaminated, dead and poorly preserved fish, close contact with people with erysipeloid, or contaminated food preparation sites (Suer andVedros, 1988; Higgins, 2000). In addi- tion, differences in pathogenicity of Erysipelothrix spp. isolates in pigs are well established (Wood et al., 1981; Takahashi et al., 1987b) and a higher susceptibility of cetaceans to certain strains should also be considered. Successful treatment of cutaneous lesions or subacute septicaemia has been reported with rapidly acting penicillin derivatives or hyperimmune serum imme- diately following the onset of clinical signs (Lacave et al., 2001). Interestingly, people exposed to infected strains isolated from fish. (A) Isolate 6 recovered from the gingival ce of a herring. (C) Isolate 12 recovered from the surface of a large . sn 370 T. Opries fish or marine environments can develop soft tissue in- fections (Finkelstein and Oren, 2011). In the present study, 10 of 14 isolates induced visible cutaneous le- sions in pigs after intradermal inoculation, further confirming the pathogenic potential of marine Erysi- pelothrix spp. isolates. Among the isolates used, serotype and spa type combinations were found to differ from terrestrial Erysipelothrix spp. isolates (Shen et al., 2010). Despite their unique antigenic properties and compositions, approximately 70% (10/14) of the marine Erysipelo- thrix spp. isolates tested were capable of inducing characteristic gross and microscopical cutaneous Fig. 3. (AeC)Section of skin from a pig infected with Erysipelothrix rhus There is focal necrosis and ulceration of the epidermis with sev There is perifollicular and circumferential multifocal suppurat ysipelothrix rhusiopathiae-positive cells (brown label) are present skin fromapig infectedwithErysipelothrix rhusiopathiae isolate 3 r of blood vessels (arrowheads) andmultifocal to focally extensive (E) Erysipelothrix rhusiopathiae-positive cells (brown) are present tion. IHC. Bar, 100 mm. (F) Section of skin from a pig infected w surface of a harbour seal. There is diffuse,moderate suppurative ig et al. lesions in pigs. It should be noted that the skin inocu- lation model is not a true model of pathogenicity, but an indication that the E. rhusiopathiae strains used can survive for at least 48 h and can cause lesions in the skin when delivered directly to that site in large num- ber. To cause disease under natural conditions may require other factors, which may or may not be pres- ent in these isolates. Conflict of Interest Statement None of the authors of this paper has a financial or personal relationship with other people or iopathiae isolate 2 recovered from the surface of a large herring. (A) ere diffuse suppurative dermatitis (arrows), HE. Bar, 150 mm. (B) ive and necrotizing folliculitis (arrows). HE. Bar, 150 mm. (C) Er- within areas of inflammation. IHC. Bar, 150 mm. (D, E) Section of ecovered from the surface of a small herring. (D)There is congestion suppurative and necrotizing dermatitis (arrows). HE. Bar 150 mm. in the epidermis and superficial dermis within areas of inflamma- ith Erysipelothrix rhusiopathiae isolate 21 recovered from the gingival epidermitis (arrow) anddermatitis (arrowhead).HE. Bar, 150mm. thrix rhusiopathiae. Microbial Pathogenesis, 25, 101e109. rapid diagnosis of swine erysipelas in formalin-fixed, ip paraffin-embedded tissue samples. Journal of Veterinary Diagnostic Investigation, 22, 86e90. Mansi W (1958) Slide gel diffusion precipitin test. Nature, 181, 1289e1290. Medway W (1980) Some bacterial and mycotic diseases of marine mammals. Journal of the American Veterinary Med- ical Association, 177, 831e834. Opriessnig T, Bender JS, Halbur PG (2010) Development and validation of an immunohistochemical method for organizations that could inappropriately influence or bias the content of the paper. Acknowledgments We thank M. Boogerd for assistance with the animal work,Dr.D.Madson for assistancewith themicroscop- ical images and the trainers and technicians of John G. Shedd Aquarium for the procurement of samples. References Bender JS, Kinyon JM, Kariyawasam S, Halbur PG, Opriessnig T (2009) Comparison of conventional direct and enrichment culture methods for Erysipelothrix spp. from experimentally and naturally infected swine. Jour- nal of Veterinary Diagnostic Investigation, 22, 86e90. Buck JD, Spotte S (1986) Microbiology of captive white- beaked dolphins (Lagenorhynchus albirostris) with com- ments on epizootics. Zoo Biology, 5, 321e329. Finkelstein R,Oren I (2011) Soft tissue infections caused by marine bacterial pathogens: epidemiology, diagnosis, and management. Current Infectious Disease Reports, 13, 470e477. Geraci JR, Sauer RM, Medway W (1966) Erysipelas in dolphins. American Journal of Veterinary Research, 27, 597e606. Grieco MH, Sheldon C (1970) Erysipelothrix rhusiopathiae. Annals of the New York Academy of Sciences, 174, 523e532. Higgins R (2000) Bacteria and fungi of marine mammals: a review. Canadian Veterinary Journal, 41, 105e116. Ingebritson AL, Roth JA, Hauer PJ (2010) Erysipelothrix rhusiopathiae: association of Spa-type with serotype and role in protective immunity. Vaccine, 28, 2490e2496. Kinsel MJ, Boehm JR, Harris B, Murnane RD (1997) Fa- tal Erysipelothrix rhusiopathiae septicemia in a captive Pa- cific white-sided dolphin (Lagenorhyncus obliquidens). Journal of Zoological and Wildlife Medicine, 28, 494e497. Lacave G, Cox E, Hermans J, Devriese L, Goddeeris BM (2001) Induction of cross-protection in mice against dol- phin Erysipelothrix rhusiopathiae isolates with a swine com- mercial vaccine. Veterinary Microbiology, 80, 247e253. Makino S, Yamamoto K, Murakami S, Shirahata T, Uemura K et al. (1998) Properties of repeat domain found in a novel protective antigen, SpaA, of Erysipelo- Pathogenicity of Marine Erys Pal N, Bender JS, Opriessnig T (2009) Rapid detection and differentiation of Erysipelothrix spp. by a novel multiplex real-time PCR assay. Journal of AppliedMicrobiology, 108, 1083e1093. Shen HG, Bender JS, Opriessnig T (2010) Identification of surface protective antigen (spa) types in Erysipelothrix reference strains and diagnostic samples by spa multi- plex real-time and conventional PCR assays. Journal of Applied Microbiology, 109, 1227e1233. Skerman VBD, McGowan V, Sneath PHA (1980) Ap- proved list of bacterial names. International Journal of Sys- temic Bacteriology, 40, 225e420. Suer LD, Vedros NA (1988) Erysipelothrix rhusiopathiae. I. Isolation and characterization from pinnipeds and bite/abrasion wounds in humans. Diseases of Aquatic Or- ganisms, 5, 1e5. Takahashi T, Fujisawa T, Benno Y, Tamura Y, Sawada T et al. (1987a) Erysipelothrix tonsillarum sp-nov isolated from tonsils of apparently healthy pigs. International Jour- nal of Systematic Bacteriology, 37, 166e168. Takahashi T, Sawada T, Muramatsu M, Tamura Y, Fujisawa T et al. (1987b) Serotype, antimicrobial suscep- tibility, and pathogenicity of Erysipelothrix rhusiopathiae isolates from tonsils of apparently healthy slaughter pigs. Journal of Clinical Microbiology, 25, 536e539. Takahashi T, Fujisawa T, Tamura Y, Suzuki S, Muramatsu M et al. (1992) DNA relatedness among Er- ysipelothrix rhusiopathiae strains representing all twenty- three serovars and Erysipelothrix tonsillarum. International Journal of Systematic Bacteriology, 42, 469e473. Takahashi T, Fujisawa T, Umeno A, Kozasa T, Yamamoto K et al. (2008) A taxonomic study on Erysi- pelothrix by DNA-DNA hybridization experiments with numerous strains isolated from extensive origins. Micro- biology and Immunology, 52, 469e478. Thurman GD, Downes SJ, Fothergill MB, Goodwin NM, HegartyMM (1983) Diagnosis and successful treatment of subacute erysipelas in a captive dolphin. Journal of the South African Veterinary Association, 54, 193e200. ToH, Nagai S (2007) Genetic and antigenic diversity of the surface protective antigen proteins of Erysipelothrix rhusio- pathiae. Clinical and Vaccine Immunology, 14, 813e820. To H, Someno S, Nagai S, Koyama T, Nagano T (2010) Immunization with truncated recombinant protein SpaC of Erysipelothrix rhusiopathiae strain 715 serovar 18 confers protective immunity against challenge with var- ious serovars. Clinical and Vaccine Immunology, 17, 1991e1997. Veraldi S, Girgenti V, Dassoni F, Gianotti R (2009) Erysi- peloid: a review. Clinical and Experimental Dermatology, 34, 859e862. Verbarg S, Rheims H, Emus S, Fruhling A, Kroppenstedt RM et al. (2004) Erysipelothrix inopinata sp. nov., isolated in the course of sterile filtration of veg- etable peptone broth, and description of Erysipelotri- chaceae fam. nov. International Journal of Systematic and Evolutionary Microbiology, 54, 221e225. WangQ, Chang BJ, Riley TV (2010)Erysipelothrix rhusiopa- thiae. Veterinary Microbiology, 140, 405e417. elothrix spp. Isolates in Pigs 371 WoodRL(1975)Erysipelothrix infection. In:DiseasesTransmit- ted from Animals to Man, WT Hubbert, WF McCullough, PR Schnurrengerger, Eds., Charles C. Thomas Limited, Springfield, pp. 271e281. Wood RL (1979) Specificity in response of vaccinated swine and mice to challenge exposure with strains of Er- ysipelothrix rhusiopathiae of various serotypes. American Journal of Veterinary Research, 40, 795e801. Wood RL, Booth GD, Cutlip RC (1981) Susceptibility of vaccinated swine and mice to generalized infection with specific serotypes ofErysipelothrix rhusiopathiae.Amer- ican Journal of Veterinary Research, 42, 608e614. Wood RL, Harrington R Jr. (1978) Serotypes of Erysipelo- thrix rhusiopathiae isolated from swine and from soil and manure of swine pens in the United States. American Journal of Veterinary Research, 39, 1833e1840. ½ Received, March 1st, 2012Accepted, August 17th, 2012 � 372 T. Opriessnig et al. Erysipelothrix rhusiopathiae Isolates Recovered from Fish, a Harbour Seal (Phoca vitulina) and the Marine Environment are C ... Introduction Materials and Methods Erysipelothrix spp. Isolates Serotyping Genotyping Spa Typing Pig Bioassay Results Characterization of Erysipelothrix spp. Strains Pathogenicity of the Erysipelothrix spp. Strains Discussion Conflict of Interest Statement Acknowledgments References