Microbiology and Immunology\nVolume 52, Issue 10 p. 469-478\nFree Access\nA taxonomic study on erysipelothrix by DNA-DNA hybridization experiments with numerous strains isolated from extensive origins\nToshio Takahashi,\nToshio Takahashi\nNational Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Kokubunji, 185-8511\nSearch for more papers by this author\nTomohiko Fujisawa,\nTomohiko Fujisawa\nLaboratory of Food Hygiene, Faculty of Applied Life Science, Nippon Veterinary and Life Science University, Musashino, 180-8602\nSearch for more papers by this author\nAnnu Umeno,\nAnnu Umeno\nSaitama Prefectural Chuo Livestock Health Station, Saitama, 331-0821\nSearch for more papers by this author\nTakashi Kozasa,\nTakashi Kozasa\nNational Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Kokubunji, 185-8511\nSearch for more papers by this author\nKinya Yamamoto,\nKinya Yamamoto\nAnimal Products Safety Division, Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Chiyoda-ku, Tokyo, 100-8950\nSearch for more papers by this author\nTakuo Sawada,\nTakuo Sawada\nLaboratory of Microbiology, Faculty of Veterinary Medicine, Nippon Veterinary and Life Sciences University, Musashino, 180-8602, Japan\nSearch for more papers by this author\nToshio Takahashi,\nToshio Takahashi\nNational Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Kokubunji, 185-8511\nSearch for more papers by this author\nTomohiko Fujisawa,\nTomohiko Fujisawa\nLaboratory of Food Hygiene, Faculty of Applied Life Science, Nippon Veterinary and Life Science University, Musashino, 180-8602\nSearch for more papers by this author\nAnnu Umeno,\nAnnu Umeno\nSaitama Prefectural Chuo Livestock Health Station, Saitama, 331-0821\nSearch for more papers by this author\nTakashi Kozasa,\nTakashi Kozasa\nNational Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Kokubunji, 185-8511\nSearch for more papers by this author\nKinya Yamamoto,\nKinya Yamamoto\nAnimal Products Safety Division, Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Chiyoda-ku, Tokyo, 100-8950\nSearch for more papers by this author\nTakuo Sawada,\nTakuo Sawada\nLaboratory of Microbiology, Faculty of Veterinary Medicine, Nippon Veterinary and Life Sciences University, Musashino, 180-8602, Japan\nSearch for more papers by this author\nFirst published: 24 September 2008\nhttps://doi.org/10.1111/j.1348-0421.2008.00061.x\nCitations: 36\nCorrespondence Toshio Takahashi, National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, 1-15-1 Tokura, Kokubunji, Tokyo 185-8511, Japan.\n\nTel: <PRESIDIO_ANONYMIZED_PHONE_NUMBER>; fax: <PRESIDIO_ANONYMIZED_PHONE_NUMBER>; email: <PRESIDIO_ANONYMIZED_EMAIL_ADDRESS>\nABSTRACT\nThe purpose of this study was to clarify the taxonomic relationship between all the serovars and species of the genus Erysipelothrix by performing DNA-DNA hybridization experiments, the customary criterion for separation of bacterial genospecies.\n\nA total of 93 strains were isolated from a wide variety of sources, including pigs affected with acute or chronic erysipelas, other diseased animals, healthy animals, fish, retail meats, and environmental materials from throughout the world during the period 1958 to 1996.\n\nThe present data on phenotypic characterization and DNA relatedness values demonstrate that 24 strains (96%) of E. tonsillarum are avirulent for swine, whereas 39 strains (66%) of genomic E. rhusiopathiae induced generalized or local urticarial lesion in swine after intradermal inoculation.\n\nThis observation suggests that genomic E. tonsillarum has little etiological significance.\n\nThree minor groups contained several strains which exhibited minimal association with each type strain of E. rhusiopathiae and E. tonsillarum.\n\nIn conclusion, it was confirmed that members of the E. rhusiopathiae and E. tonsillarum groups resemble each other in regard to many phenotypic characteristics, but differ in their ability to produce acid from saccharose and in their pathogenicity for swine.\n\nThe genus Erysipelothrix certainly contains two main species: E. rhusiopathiae and E. tonsillarum.\n\nList of Abbreviations:\nOrganisms of the genus Erysipelothrix, particularly E. rhusiopathiae, have a major economic impact, causing a variety of diseases in animals and birds (ranging from septicemia to urticaria), as well as erysipeloid or mitro-aortic infective endocarditis in humans (1, 2).\n\nThese organisms have also been isolated from tonsils of apparently healthy slaughter swine, from slime on fish, and from the environment.\n\nIn general, strains of Erysipelothrix have been classified into 23 serovars and type N strains, which produce no precipitating antibody against homologous and heterologous heat-stable extracts in rabbits (3).\n\nMost isolates obtained from diseased swine belong to serovars 1a, 1b, and 2 (2, 4, 5).\n\nThe genus Erysipelothrix had been thought to contain a single species, E. rhusiopathiae, despite its wide range of hosts and wide variety of serological characteristics over many years (6).\n\nHowever, a cluster of avirulent serovar 7 strains which were isolated from tonsils of apparently healthy slaughter pigs were found to be genetically distinct from other E. rhusiopathiae strains.\n\nThese strains have previously been reported by us to be a new species, Erysipelothrix tonsillarum (7, 8).\n\nThe type strain of the new species, E. tonsillarum, is ATCC43339 of serovar 7 isolated from porcine tonsil (8).\n\nMoreover, on the basis of the concentrations of DNA common among the 23 serovar reference strains (one strain stipulated internationally for each serovar), it was reported by us that the genus Erysipelothrix contains two species, E. rhusiopathiae (including serovars 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 17, 19, 21 and type N) and E. tonsillarum (serovars 3, 7, 10, 14, 20, 22 and 23) (9).\n\nOur previous investigations (7–9) suggested that E. tonsillarum could be distinguished from E. rhusiopathiae mainly by serology.\n\nSome investigators have proposed that serotyping was a somewhat questionable method for identifying isolates as either E. rhusiopathiae or E. tonsillarum, their arguments being based on results of analyses of field isolates and some serovar reference strains of Erysipelothrix by multilocus enzyme electrophoresis (10) and ribotyping (11),.\n\nOn the basis of these reports on phenotypic and ribotypic properties, some isolates belonging to the same serovar might be considered to be different species, but whether they should be classified as genomic E. rhusiopathiae or E. tonsillarum was uncertain.\n\nThe purpose of this study was to clarify the taxonomic relationship between all serovars and species of the genus Erysipelothrix by performing DNA-DNA hybridization experiments, the customary criterion for separation of bacterial genospecies, on a large collection of isolates from extensive origins in each serovar.\n\nMATERIALS AND METHODS\nBacterial strains\nDetails concerning the test strains and their sources are shown in Table 1.\n\nA total of 93 strains were isolated from a wide variety of sources including pigs affected with acute or chronic erysipelas, other diseased animals, healthy animals, fish, retail meats, and environmental materials from throughout the world during the period 1958 to 1996.\n\nStrains E. rhusiopathiae ATCC 19414T and E. tonsillarum ATCC 43339T were obtained from the American Type Culture Collection, Rockville, MD, USA.\n\nThe strains were maintained on stock culture agar (Difco Laboratories, Detroit, MI, USA).\n\nTable 1.\n\nSerovars, origins, sources, and levels of pathogenicity for mice and swine of Erysipelothrix strains\nStrain or isolate designation † Serovar Year of isolation Origin Source Pathogenicity for mice(logLD50) ‡ Pathogenicity for swine § Size of erythema (cm) Systemic ¶ ME-7* 1a Unknown Unknown Unknown 1.0 Generalized + Sayo-SP 1a 1979 Hyogo, Japan Chicken with septicemia 1.1 Generalized + Kanagawa96-1 1a 1995 Kanagawa, Japan Pig with septicemia 1.9 Generalized + YamagataFD 1a 1995 Yamagata, Japan Duck with septicemia 1.5 Generalized + 422/1E1* 1b 1958 United States Porcine spleen 1.0 Generalized + NKB-105 1b 1994 Tokyo, Japan Boar meat 1.9 Generalized + 82-517 1b 1982 Tokyo, Japan Pig with arthritis 1.9 Generalized + ATCC19414 T 2 Unknown Unknown Pig with endocarditis 1.4 Generalized + R32E11* 2 Unknown Unknown Unknown 1.5 Generalized + NF4E1* 2 1958 United States Porcine spleen 1.3 Generalized + K-209 2 1996 Kanagawa, Japan Pig with arthritis 1.7 Generalized + Kohriyama5-41 2 1993 Fukushima, Japan Pig with urticaria 2.1 – – Witlling* 3 Unknown Unknown Fish 0.5 – – Tama94-160 3 1994 Tokyo, Japan Chicken meat 0.6 – – NKF-245 3 1993 Tokyo, Japan Fish 1.6 – – NKF-403 3 1993 Tokyo, Japan Fish 1.1 – – NKF-246 3 1993 Tokyo, Japan Fish 1.6 – – NKF-247 3 1993 Tokyo, Japan Fish 1.8 – – NKF-402 3 1993 Tokyo, Japan Fish 1.6 – – Doggerscharbe 4 1958 Germany Fish >7.0 – – NKF-425 4 1993 Tokyo, Japan Fish 1.6 13 × 15 – NKF-336 4 1993 Tokyo, Japan Fish 1.5 – – Pécs 67* 5 1970 Hungary Porcine tonsil 1.4 15 × 17 – Dolphin S-1 5 1982 Yamaguchi, Japan Dolphin with septicemia 2.2 – – S-101 5 1996 Kanagawa, Japan Pork 2.2 – – Agata 5 Unknown Unknown Pig with septicemia 2.0 – – Tuzok* 6 1970 Hungary Bustard 0.8 – – Dolphin E-1 6 1980 Kanagawa, Japan Dolphin with septicemia 0.4 – – S-146 6 1996 Kanagawa, Japan Pig with arthritis 2.3 – – NKB-2 6 1994 Tokyo, Japan Boar meat 1.9 – – ATCC43339 T 7 1984 Tokyo, Japan Porcine tonsil 5.3 – – ATCC43338 7 1984 Tokyo, Japan Porcine tonsil 5.7 – – P-43* 7 1960 Japan Fish 1.8 – – L1-3 7 1988 Tokyo, Japan Pork 0.9 – – L1-4 7 1988 Tokyo, Japan Pork 0.8 – – Nairiku-21 7 1986 Yamagata, Japan Pig with lymphadenitis 0.8 – – NG-37 7 1986 Nagasaki, Japan Pig slurry 1.0 – – T-131 7 1972 Germany Porcine tonsil 1.2 – – Kanagawa-7 7 1989 Kanagawa, Japan Pig with arthritis 1.9 – – NKB-13 7 1994 Tokyo, Japan Boar meat 1.2 – – NKB-41 7 1994 Tokyo, Japan Boar meat 1.4 – – NKB-183 7 1994 Tokyo, Japan Boar meat 1.6 – – NKB-199 7 1994 Tokyo, Japan Boar meat 3.5 – – 266/719 7 Unknown Belgium Dog with endocarditis 1.3 – – 266/6171b 7 Unknown Belgium Dog with endocarditis 1.0 – – 266/6969 7 Unknown Belgium Dog with endocarditis 0.7 - – 266/7946 7 Unknown Belgium Dog with endocarditis 1.1 – – 266/7991 7 Unknown Belgium Dog with endocarditis 1.3 – – Goda* 8 1970 Hungary Godwit 0.6 10 × 10 – NKF-263 8 1993 Tokyo, Japan Fish 1.7 – – 82-911 8 1982 Tokyo, Japan Pig with lymphadenitis 1.6 6 × 6 – Kaparek* 9 1962 Hungary Fish >6.9 – – IT-401 9 1987 Indonesia Porcine tonsil 1.8 8 × 9 – IT-349 9 1987 Indonesia Porcine tonsil 1.5 7 × 8 – IT-497 9 1987 Indonesia Porcine tonsil 2.0 – – 14B 9 1987 United States Pen soil 1.8 15 × 15 – Lengyel-P* 10 Unknown Unknown Squirrel 0.2 – – 2179 10 1977 United States Pig slurry 2.0 Generalized – IV12/8* 11 1964 Hungary Porcine tonsil 0.9 16 × 17 – K-503 11 1996 Kanagawa, Japan Pig with arthritis >7.8 – – NKB-60 11 1994 Tokyo, Japan Boar meat 2.0 8 × 9 – Pécs 9* 12 1970 Hungary Porcine tonsil 0.7 Generalized + NKF-1 12 1993 Tokyo, Japan Fish >7.4 – – Iida-4 12 1987 Tokyo, Japan Pig with lymphadenitis 1.9 7 × 7 – Pécs 56* 13 1970 Hungary Porcine tonsil >6.5 – – Pécs 18 13 1974 Hungary Porcine tonsil >8.6 – – Shiribeshi-17 13 1989 Hokkaido, Japan Pig slurry >7.4 – – Shiribeshi-19 13 1989 Hokkaido, Japan Pig slurry >7.1 – – Iszap-4* 14 1970 Hungary Mud of zoo pond 0.8 – – Pécs 3597* 15 1971 Hungary Porcine tonsil 1.2 – – NKF-206 15 1993 Tokyo, Japan Fish >6.9 – – 2909-H 15 1990 Yamagata, Japan Pig with endocarditis 1.9 – – 2909-S 15 1990 Yamagata, Japan Pig with endocarditis 2.0 9 × 9 – Tanzania* 16 1973 Hungary Parrot 1.4 – – NKD-96 16 1994 Tokyo, Japan Deer meat 1.8 – – NKB-97 16 1994 Tokyo, Japan Boar meat 1.4 – – T-184 16 1984 Tokyo, Japan Porcine tonsil 0.5 6 × 10 – 545* 17 1971 United States Porcine spleen >6.5 – – S-106 17 1996 Kanagawa, Japan Pork >7.7 – – Shizuoka-55 17 1983 Shizuoka, Japan Pig with arthritis >8.7 – – 715* 18 1971 United States Porcine spleen 1.1 18 × 20 – 2017* 19 1972 United States Porcine spleen 0.9 12 × 16 – NKB-78 19 1994 Tokyo, Japan Boar meat 1.7 – – NKD-82 19 1994 Tokyo, Japan Deer meat 1.8 5 × 6 – 2553* 20 1975 United States Porcine spleen 1.5 10 × 12 – Bano 36* 21 Unknown Unknown Sheep dip 0.8 14 × 18 – K-303 21 1996 Kanagawa, Japan Pig with arthritis 2.0 12 × 15 – 82-533 21 1982 Tokyo, Japan Pig with arthritis 2.0 – – Bano 107* 22 Unknown Unknown Sheep dip >5.9 – – KS20A* 23 Unknown Unknown Pig slurry 1.4 – – MEW22* N 1958 United Kingdom Unknown 1.0 – – Shizuoka-12 N 1982 Shizuoka, Japan Pig with lymphadenitis 2.0 14 × 15 – 82-506 N 1982 Tokyo, Japan Pig with arthritis >8.6 – –\nCultural and biochemical tests\nThe strains were characterized biochemically using the tests listed in Table 2.\n\nProduction of H2S was tested in triple sugar iron agar (Difco).\n\nCytochrome oxidase test strips (Eiken Chemical, Tokyo, Japan) were used to detect the presence of this enzyme.\n\nPlasma-clotting activity was detected by mixing a 24-hr broth culture with fresh citrated rabbit plasma (12).\n\nAll the other biochemical test procedures used by us have been described previously (8).\n\nThe medium used to test for production of acid from carbohydrates was nutrient broth supplemented with 1% Andrade's indicator (self-preparation) and 10% horse serum (Gibco, Carlsbad, CA, USA) (13).\n\nAntimicrobial susceptibility was tested using an agar dilution method (14).\n\nTable 2.\n\nDifferential phenotypic characteristics of the Erysipelothrix species\nCharacteristic % of strains having a positive reaction E. rhusiopathiae (59 strains) E. tonsillarum (25 strains) Other sp.-1 (4 strains) Other sp.-2 (3 strains) Other sp.-3 (2 strains) H 2 S production (triple sugar iron agar) 100 100 100 100 100 Test tube brush in gelatin 100 100 0 100 100 Catalase activity 0 0 0 0 0 Oxidase activity 0 0 0 0 0 Acid produced from: Saccharose 0 96 0 0 0 Glycerol 0 0 0 0 0 Maltose 100 100 100 100 100 Salicin 0 8 100 0 50 Inosite 0 0 0 0 0 Galactose 100 100 50 100 100 Levulose 97 100 100 100 100 Dulcitol 0 0 0 0 0 Rhaffinose 0 0 0 0 0 Mannite 0 0 0 0 0 Mannose 85 20 100 100 0 Lactose 100 100 0 100 100 Glucose 100 100 100 100 100 Rhamnose 0 0 0 0 0 Treharose 0 0 100 0 0 Inulin 0 0 0 0 0 Xylose 0 0 0 0 0 Sorbitol 0 0 0 0 0 Arabinose 0 0 0 0 0 Clotting of citrated plasma 98 100 75 100 100 Susceptibility to: Penicillin G (1 U/ml) 100 100 100 100 100 Ampicillin (1 μg/ml) 100 100 100 100 100 Kanamycin (100 μg/ml) 0 0 0 0 0 Sulfadimethoxine (100 μg/ml) 0 0 0 0 0 Pathogenicity for swine: Generalized erythema 39 0 0 0 0 Localized erythema 27 4 0 100 0 No clinical signs 34 96 100 0 100\nTest used to determine serovars\nThe serovars of the strains were determined by using a previously described method (15–17).\n\nColonies from a 48-hr agar plate of each strain were inoculated into tryptose phosphate broth (Difco, pH7.6) containing 0.1% Tween 80 (TPBT).\n\nAfter incubation for 48 hr at 37°C, the broth culture was centrifuged at 12 000 rpm for 20 min.\n\nThe bacteria were then washed three times with physiological saline and suspended in distilled water to 1/30 of the original volume.\n\nThe resulting bacterial suspension was autoclaved for one hr at 121°C, cooled, and clarified by centrifugation.\n\nThe supernatant fluid was tested using an agar gel double-diffusion precipitation system against typing sera (rabbit sera) representing serovars 1a, 1b, 2 through 23 and type N of Erysipelothrix.\n\nPathogenicity test\nFor pathogenicity tests, four week old female outbred strain ddY mice (Nippon SLC, Hamamatsu, Japan) were used.\n\nFemale and castrated male Yorkshire swine purchased from the Minano Agricultural Cooperative Association for Laboratory Animals, Saitama, Japan were used when they were three to four months old.\n\nThese swine were conventionally farrowed and raised in confinement.\n\nThe sera of the swine had growth agglutination titers (18) of <1:8.\n\nThe levels of strain pathogenicity for mice and swine were determined by using a previously described method (8).\n\nA portion (0.1 ml) of one of a series of 10-fold dilutions of a TPBT culture of each strain was injected subcutaneously into each of five mice.\n\nAt same time, 0.1 ml of each dilution was spread onto two Petri dishes with mixed heart infusion broth (Eiken) containing 0.75% agar.\n\nAfter 48 hr of cultivation at 37°C, the colonies in the agar medium were counted.\n\nTo determine the LD50, mortality rates were recorded 14 days after exposure.\n\nLD50 values were determined by using the method of Karber (19).\n\nOne pig was inoculated intradermally with 0.1 ml of a TPBT culture (approximately 107 CFU per pig) of each strain.\n\nClinical signs were observed every day for 14 days after exposure.\n\nAll animal studies were conducted in accordance with the National Veterinary Assay Laboratory Guide for the Care and Use of Laboratory Animals, and the relevant Animal Welfare Acts.\n\nDetermination of enzymatic activities\nThe API ZYM system (API System SA, La Balme les Grottes, Montalieu-Vercieu, France) was used to detect the presence of 19 constitutive enzymes by chromogenic substrates according to a previously described method (20).\n\nPreparation of DNA\nFor the preparation of DNA, bacterial cells grown in TPBT were harvested in logarithmic phase and washed twice with 0.15M NaCl–0.1M EDTA (pH 8.0).\n\nThe DNA was isolated by using a modification of the procedure of Marmur (21).\n\nThe purity and amount of DNA were estimated by measuring the hyperchromic shift during thermal denaturation (22).\n\nTritium-labeled DNA was prepared by using a nick translation system (New England Nuclear, Boston, MA, USA) adapted from the procedure of Rigby et al.\n\n(23).\n\nDNA homology experiments\nDNA homology experiments were performed using the S1 nuclease procedure as described by Johnson et al.\n\n(24).\n\nRESULTS\nThe results of pathogenicity tests are shown in Table 1.\n\nIn swine, thirteen strains belonging to serovars 1a, 1b, 2, 10 and 12 induced generalized urticarial lesions after intradermal inoculation.\n\nNineteen strains belonging to serovars 4, 5, 8, 9, 11, 12, 15, 16, 18 through 21, and N induced local urticarial lesion at the site of inoculation.\n\nThe remaining strains, which belonged to serovars 3 through 17, 19, 21 through 23, and N, induced no clinical signs.\n\nOf the 93 strains tested, 76 (belonging to serovars 1a through 12, 14 through 16, 18 through 21, and 23 through N) were highly virulent for mice (LD50 values, ≤102.2 CFU).\n\nOnly one strain, belonging to serovar 7, (NKB-199 originating from boar meat) was weakly virulent (LD50 values, 103.5 CFU).\n\nThe remaining 16 strains (belonging to serovars 4, 7, 9, 11 through 13, 15, 17, 22, and N) had LD50 values of more than 105.3 CFU.\n\nThe DNA base composition of strains tested and results of DNA-DNA hybridization experiments are shown in Table 3.\n\nThe strains had guanine-plus-cytosine contents ranging from 35 to 40 mol% of DNA but separated into five genomic groups on the basis of the percentages of DNA-DNA re-association with the type strains of E. rhusiopathiae and E. tonsillarum.\n\nThe E. rhusiopathiae strains belonging to serovars 1a, 1b, through 2 to 6, 8, 9, 11, 12, 15 through 17, 19, 21, and N hybridized at levels of 61% or more with the type strain of that species, and the E. tonsillarum strains belonging to 3, 7, 10, 14 through 16, 20, 22, and 23 hybridized at levels of 66% or more with the type strain of E. tonsillarum.\n\nIn each case the non-homologous values were <37%.\n\nThere were three minor groups (other sp.-1, other sp.-2, and other sp.-3) which exhibited low levels of hybridization (4 to 47%) with each type strain of E. rhusiopathiae and E. tonsillarum.\n\nA reorganization of taxonomic classifications within the Erysipelothrix species based on these findings is described in Table 4.\n\nAlthough most of strains tested were differentiated into the inherent serovar as reported previously (9), thirteen strains (14%) of genomic E. rhusiopathiae, E. tonsillarum, other sp-2, and other sp-3 occurred in common serovars 3 (six strains), 7 (two strains), 9 (one strain), 10 (one strain), 15 (one strain) and 16 (two strains).\n\nTable 3.\n\nLevels of DNA relatedness among E. rhusiopathiae and E. tonsillarum strains\nStrain or isolate designation † Serovar Guanine-plus-cytosine content of DNA (mol%) %Homology with [ 3 H] DNA from: Strain ATCC19414 T Strain ATCC43339 T E. rhusiopathiae ATCC19414 T 2 38 100 25 ME-7* 1a 36 89 14 Sayo-SP 1a 37 97 16 Kanagawa96-1 1a NT 102 19 YamagataFD 1a NT 96 31 422/1E1* 1b 37 92 24 NKB-105 1b NT 103 15 82-517 1b NT 76 10 R32E11* 2 40 97 18 NF4E1* 2 37 102 37 K-209 2 NT 69 16 Kohriyama5-41 2 NT 70 11 Tama94-160 3 NT 81 22 NKF-245 3 NT 78 26 NKF-403 3 NT 64 19 NKF-246 3 NT 94 22 NKF-247 3 NT 64 13 NKF-402 3 NT 63 13 Doggerscharbe 4 36 76 17 NKF-425 4 NT 80 17 NKF-336 4 NT 66 12 Pécs 67* 5 37 99 21 Dolphin S-1 5 37 86 21 S-101 5 NT 100 21 Agata 5 NT 78 14 Tuzok* 6 39 73 20 Dolphin E-1 6 37 77 17 S-146 6 NT 76 25 NKB-2 6 NT 93 18 Goda* 8 36 101 18 NKF-263 8 NT 98 21 82-911 8 NT 86 13 Kaparek* 9 37 80 12 IT-349 9 NT 67 14 IT-497 9 NT 61 11 14B 9 NT 68 11 IV12/8* 11 36 89 15 K-503 11 NT 91 19 NKB-60 11 NT 66 12 Pécs 9* 12 37 87 14 NKF-1 12 NT 95 34 Iida-4 12 NT 61 11 Pécs 3597* 15 38 80 12 2909-H 15 NT 103 16 2909-S 15 NT 107 13 Tanzania* 16 36 82 11 T-184 16 36 68 15 545* 17 37 96 17 S-106 17 NT 86 18 Shizuoka-55 17 NT 77 16 2017* 19 39 91 16 NKB-78 19 NT 92 16 NKD-82 19 NT 62 15 Bano 36* 21 40 85 17 K-303 21 NT 88 21 82-533 21 NT 85 11 MEW22* N 40 93 21 Shizuoka-12 N NT 101 25 82-506 N NT 76 12 E.tonsillarum ATCC43339 T 7 35 23 100 Witlling* 3 37 16 94 ATCC43338 7 37 15 103 P-43* 7 36 20 87 L1-3 7 38 24 82 L1-4 7 38 21 79 Nairiku-21 7 38 23 66 NG-37 7 36 24 77 T-131 7 37 18 75 Kanagawa-7 7 NT 10 105 NKB-13 7 NT 11 103 NKB-41 7 NT 14 104 266/719 7 NT 12 81 266/6171b 7 NT 13 103 266/6969 7 NT 11 92 266/7946 7 NT 12 104 266/7991 7 NT 11 83 Lengyel-P* 10 37 26 78 Iszap-4* 14 36 21 80 NKF-206 15 NT 21 101 NKD-96 16 NT 14 86 NKB-97 16 NT 18 78 2553* 20 36 26 74 Bano 107* 22 38 27 80 KS20A* 23 38 26 81 Other sp.-1 Pécs 56* 13 35 24 19 Pécs 18 13 NT NT NT Shiribeshi-17 13 35 13 12 Shiribeshi-19 13 36 12 8 Other sp.-2 715* 18 38 47 16 IT-401 9 NT 23 11 2179 10 NT 26 4 Other sp.-3 NKB-183 7 NT 18 26 NKB-199 7 37 17 14\nTable 4.\n\nReorganization of taxonomic classification within Erysipelothrix species\nErysipelothrix species Serovars included in the species Number of strains examined %Homology with [ 3 H] DNA from: Strain ATCC19414 T Strain T ATCC43339 T Strain Pécs 56 Strain 715 Strain NKB-199 E. rhusiopathiae 1a, 1b, 2, 3, 4, 5, 6, 8, 9, 11, 12, 15, 16, 17, 19, 21, N 59 >62 <37 15 † 38 † 18 † E. tonsillarum 3, 7, 10, 14, 15, 16, 20, 22, 23 25 <27 >66 13 ‡ 23 ‡ 22 ‡ Other sp.-1 13 4 <24 <19 >86 <10 <14 Other sp.-2 9, 10, 18 3 <47 <16 <13 >80 <24 Other sp.-3 7 2 <18 <26 4 § 31 § >100\nThe differential biochemical and pathogenic characteristics of the Erysipelothrix species are shown in Table 2.\n\nApart from the ability to produce acid from saccharose, the biochemical characteristics of the major Erysipelothrix species were identical; 24(96%) of 25 strains of E. tonsillarum produced acid from saccharose, whereas none of the 59 strains of E. rhusiopathiae did so.\n\nAll four strains belonging to other sp.-1 differed clearly from those of the other Erysipelothrix species in their ability to produce acid from salicin and treharose, and in test tube brush growth in gelatin.\n\nIn the pathogenicity test, 39 (66%) of the 59 strains of E. rhusiopathiae induced generalized or localized erythema in swine after intradermal inoculation, whereas 24 (96%) of the 25 strains of E. tonsillarum did not induce any clinical sign of erysipelas in swine.\n\nThe enzymatic profiles of the Erysipelothrix species are shown in Table 5.\n\nThe profiles of these five genomic species were very similar, differing solely in α-glucosidase activity of other sp.-1, as well as the ability to produce acid from carbohydrates.\n\nTable 5.\n\nDifferential enzymatic activities of the Erysipelothrix species\nEnzyme assayed % of strains having a positive reaction E. rhusiopathiae (59 strains) E. tonsillarum (25 strains) Other sp.-1 (4 strains) Other sp.-2 (3 strains) Other sp.-3 (2 strains) Alkaline phosphatase 54 16 50 100 100 Butyrate esterase 0 0 50 33 0 Caprylate esterase(C8) 24 36 100 100 0 Myristate lipase(C4) 0 0 0 0 0 Leucine arylamidase 86 80 0 67 100 Valine arylamidase 5 0 0 0 0 Cystine arylamidase 25 4 0 33 50 Trypsin 0 0 0 0 0 Chymotrypsin 83 100 100 67 100 Acid phosphatase 100 100 100 100 100 Phosphamidase 97 96 100 100 100 α-Galactosidase 0 0 0 0 0 β-Galactosidase 0 0 0 0 0 β-Glucuronidase 0 0 0 0 0 α-Glucosidase 0 0 100 0 0 β-Glucosidase 0 0 0 0 0 N-Acetyl-β-glucosaminidase 93 76 0 100 0 α-Mannosidase 0 0 0 0 0 α-Fucosidase 0 0 0 0 0\nDISCUSSION\nThe present data on phenotypic characterization and DNA-DNA hybridization experiments with numerous strains isolated from extensive origins demonstrated that 24 strains (96%) of the genomic E. tonsillarum were avirulent for swine except for strain 2553 of serovar 20 isolated from porcine spleen, which induced a local urticarial lesion at the site of inoculation, whereas 39 strains (66%) of the genomic E. rhusiopathiae induced generalized or local urticarial lesions after intradermal inoculation.\n\nThis observation, in conjunction with previously published epidemiological data obtained by examining serovars of a large number of clinical isolates (1046 strains) from a wide area of Japan over the past 11 years (5), suggests that E. tonsillarum has little etiological significance.\n\nIt is well known that E. rhusiopathiae causes a variety of diseases in a wide range of animal hosts.\n\nA previous study by us (25) of experimental infection with the serovar reference strains of Erysipelothrix and strain Sayo-SP (used in the present study) of serovar 1a isolated from chickens with septicemia confirmed that strains of some serovars of E. rhusiopathiae are pathogenic for chicken.\n\nHowever in that study E. tonsillarum strains were not found to be pathogenic, indicating the need to omit E. tonsillarum as a possible causal organism for erysipelas in chickens, as is the case for swine erysipelas.\n\nOn the other hand, all isolates studied from dogs with endocarditis in Belgium were found by us to belong to serovar 7; they produced acid from saccharose but did not induce any clinical signs in swine (26).\n\nSubsequently, it was definitely verified by us that all canine strains belonging to serovar 7 hybridize at levels of 81% or more with the type strain of E. tonsillarum, but have little DNA homology (11 to 13%) with the type strain of E. rhusiopathiae.\n\nBased on DNA-DNA hybridization results it was confirmed that at least serovar 7 of isolates from dogs with endocarditis are definitely identifiable as E. tonsillarum (27).\n\nThese results, taken in conjunction with previous clinical case reports (28–30), strongly indicate that some strains of genomic E.tonsillarum are canine pathogens.\n\nChooromoney et al.\n\n(10) have reported that the Australian reference strain for serovar 7 is genetically typical of E. rhusiopathiae in the multilocus enzyme electrophoretic patterns of 74 field isolates and 22 reference strains for serovars of Erysipelothrix.\n\nAdditionally, by determination of restriction fragment length polymorphisms of 16S rRNA gene (also called ribotyping) of 32 Erysipelothrix strains, strains of serovar 7 were found to have different ribopatterns with similarity distances of approximately 75 (11).\n\nMultilocus enzyme electrophoresis and ribotyping have been proven to be useful taxonomic or epidemiological techniques for examining the genetic structure or intraspecies variations of a large collection of bacteria (31, 32).\n\nThe present study using DNA-DNA hybridization, the customary criterion for separation of bacterial genospecies, also suggests that serotyping of Erysipelothrix is not necessarily an absolute or essential taxonomic tool.\n\nOf 16 field strains (except for the type strain [ATCC 43339T] and the reference strain [ATCC 43338T] belonging to serovar 7 of the representative serovar in E. tonsillarum) used in the present investigation, 14 strains (88%) could certainly be classified as genomic E. tonsillarum, indicating that determination of at least serovar 7 could be a possible method for identifying isolates as E. tonsillarum.\n\nVery recently Erysipelothrix isolated in the course of sterile filtration of vegetable peptone broth has been proposed to constitute a new species, E.inopinata sp.\n\nnov., based on 16S rRNA gene sequence comparison (33).\n\nThe detailed phenotypic characteristics, including serovar, of this new species are still unclear.\n\nThe low DNA relatedness values (4 to 47%) between the nine field strains and the type strains of the two major species indicate that the three minor groups (other sp.-1 [serovar 13], other sp.-2 [serovar 9, 10, and 18], and other sp.-3 [serovar 7]) may be members of a new genomic species of the genus Erysipelothrix.\n\nThis possibility is supported by differences in their ability to produce acid from carbohydrates, and in enzymatic activities, as compared to the major Erysipelothrix species.\n\nHowever, more strains will have to be collected to support this hypothesis.\n\nIn any case, further DNA-DNA hybridization studies with the type strain DSM 15511T of E. inopinata would be needed to clarify the taxonomic relationships between the three minor groups and E. inopinata.\n\nBased on our present study with a large collection of isolates from extensive origins in each serovar, it was concluded that the members of the E. rhusiopathiae and E. tonsillarum groups resemble each other in many phenotypic characteristics, but differ in their ability to produce acid from saccharose and in their pathogenicity for swine, and that the genus Erysipelothrix certainly contains two main species, E. rhusiopathiae and E. tonsillarum.\n\n----------\nAs a professional microbiologist, create a concise and comprehensive Chinese scientific report on Erysipelothrix Spp. research based on the provided text, whether it's an article, post, conversation, or passage. Follow these guidelines:\n\n 1. Produce a detailed and in-depth report that remains clear and concise.\n 2. Focus on main ideas and essential information, removing any extraneous language.\n 3. Strictly rely on the provided text without adding external information.\n 4. Format the report in paragraphs for easy understanding.\n 5. Conclude with “[End of Notes, Message #X],” where “X” represents the total number of messages I’ve sent. Increment the message counter with each new message.\n 6. Use English for technical terms, industry terminology, proper nouns, and species names. After you finish, add the following words: {{{ I_finished_papa!!! }}} to the end of you answer