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DNA relatedness among Erysipelothrix rhusiopathiae strains representing all twenty-three serovars and Erysipelothrix tonsillarum.

The levels of relatedness among strains of Erysipelothrix rhusiopathiae (serovars 1 through 23 and type N) were estimated by performing DNA-DNA hybridization experiments with the type strains of E. rhusiopathiae and Erysipelothrix tonsillarum, which are the two Erysipelothrix species that have been described. Two distinct DNA relatedness groups were identified. The group 1 strains, representing serovars 1, 2, 4 through 6, 8, 9, 11, 12, 15 through 17, 19, and 21 and type N, exhibited more than 73% hybridization with the type strain of E. rhusiopathiae but less than 24% hybridization with the type strain of E. tonsillarum. Group 2 included serovar 3, 7, 10, 14, 20, 22, and 23 strains, and these strains exhibited more than 66% hybridization with the type strain of E. tonsillarum but less than 27% hybridization with the type strain of E. rhusiopathiae. Strains representing serovars 13 and 18 exhibited low levels of hybridization (16 to 47%) with both of the type strains, indicating that these serovars may be members of a new genomic species. The members of the E. rhusiopathiae and E. tonsillarum groups resembled each other in many phenotypic characteristics, but differed in their ability to produce acid from saccharose and in their pathogenicity for swine. Our results confirm that the genus Erysipelothrix contains two main genomic species, E. rhusiopathiae and E. tonsillarum, which can be differentiated into serovars.

Animals↗

A multiplex polymerase chain reaction for discriminating Erysipelothrix rhusiopathiae from Erysipelothrix tonsillarum.

Erysipelothrix rhusiopathiae is the causative agent of swine erysipelas, and it causes great economic losses in Japan and worldwide. In meat inspection, it is very important to distinguish E. rhusiopathiae from other bacteria showing similar clinical signs of disease or similar bacterial characteristics. To distinguish E. rhusiopathiae from Erysipelothrix tonsillarum, 2 polymerase chain reaction (PCR) systems were combined. The primer sets ERY-1F and ERY-2R were designed to amplify 2210 base pairs (bp) of nucleotide sequence specific for E. rhusiopathiae chromosomal DNA, and the primer sets MO101 and ERS-1R were designed to amplify 719 bp of nucleotide sequence including a highly conserved region of genus Erysipelothrix 16S rRNA. Two fragments were amplified when E. rhusiopathiae was used as the PCR template using the primer sets, whereas a single fragment was amplified when E. tonsillarum was used as the template. No fragments were amplified when nucleic acid from other bacteria that cause clinical signs similar to swine erysipelas were used as the template. Moreover, 5 specimens collected from postinspected swine carcasses were diagnosed as E. rhusiopathiae using the PCR described in this study, in agreement with results of microbiological tests for the genus Erysipelothrix, whereas negative samples were negative both in conventional bacterial tests and by PCR. The detection limit of multiplex PCR ranged from 10(2) to 10(4) colony forming units per reaction tube for positive samples. These results suggest that this method is useful for screening of swine erysipelas in meat inspection centers.

Animals↗

Taxonomic evidence that serovar 7 of Erysipelothrix strains isolated from dogs with endocarditis are Erysipelothrix tonsillarum.

The levels of relatedness among strains of Erysipelothrix serovar 7 isolated from dogs with endocarditis were estimated by performing DNA-DNA hybridization experiments with the type strains of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum. All the canine strains exhibited more than 81% hybridization with the type strain of E. tonsillarum but less than 13% hybridization with the type strain of E. rhusiopathiae. Based on DNA-DNA hybridization results we confirmed that serovar 7 of the isolates from dogs with endocarditis were conclusively identified as E. tonsillarum. These results strongly indicate that some strains of genomic E. tonsillarum are a canine pathogen.

Animals↗

Differentiation of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell proteins.

The protein patterns of whole cells of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum were studied by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein patterns of the 16 strains of E. rhusiopathiae and E. tonsillarum studied, including the type strains of these two species, resembled each other, except that there were 71-, 41-, 34-, and 26-kDa proteins in the E. rhusiopathiae pattern and 74-, 44-, 36-, and 25-kDa proteins in the E. tonsillarum pattern. This observation indicates that there is some phenotypic heterogeneity in the genus Erysipelothrix. In addition, the protein patterns of E. rhusiopathiae serotype reference strains representing serotypes 1 through 23 and type N were compared. The protein patterns of serotype 1a, 1b, 2, 4, 5, 6, 8, 9, 11, 12, 15, 16, 19, and 21 and type N strains were similar to the pattern of the type strain of E. rhusiopathiae (strain ATCC 19414). Conversely, the protein patterns of serotype 3, 7, 10, 14, and 20 strains were very similar to the pattern of the type strain of E. tonsillarum (strain ATCC 43339). An atypical pattern was observed in serotype 13, 17, 18, 22, and 23 strains. These results suggest that this method may be used as an aid in studying the taxonomy of these bacteria.

Animals↗

Computerized comparison of the protein compositions of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum strains.

Protein profiles of six Erysipelothrix rhusiopathiae strains, five Erysipelothrix tonsillarum strains and three Erysipelothrix strains of uncertain taxonomic position were studied by sodium dodecyl sulphate-polyactylamide gel electrophoresis (SDS-PAGE). In a computerized comparison of the protein patterns of the strains, the level of similarity between the strains was determined. The SDS-PAGE protein bands were divided into 14 groups based on molecular weight. The relative distribution of proteins within these groups was used to characterize the strains. These distribution patterns were analysed by computing Pearson's correlation coefficient between strains, and by cluster analysis based on Euclidean distances and the unweighted pair-group method of arithmetic averages (UPGMA). The geometric mean of the similarities calculated by Pearson's correlation coefficient was 0.980 +/- 0.018 between the E. rhusiopathiae strains and 0.979 +/- 0.013 for E. tonsillarum strains. The value was 0.932 +/- 0.036 between the strains belonging to different species. However, a threshold value applicable for identification of a given strain to a species could not be established. Of the three strains of uncertain taxonomic position, the strains designated Rotzunge and Iszap 4 had a protein composition more similar to that of E. tonsillarum than to that of the E. rhusiopathiae type strain. The strain designated Pécs 56, which may be a member of a new species according to literature data, gave inconsistent results by the two methods used. The computerized evaluation method developed here is suitable for the comparison of the protein composition of the strains and for the construction of the protein similarity tree by cluster analysis.

Animals↗

Analysis of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum by multilocus enzyme electrophoresis.

The genetic diversity of 74 Australian field isolates of Erysipelothrix rhusiopathiae and 22 reference strains for serovars of E. rhusiopathiae or Erysipelothrix tonsillarum was examined by multilocus enzyme electrophoresis. Four serovar reference strains of E. tonsillarum (strains KS 20 A, Wittling, Lengyel-P, and Bano 107 for serovars 25, 3, 10, and 22, respectively) were genetically distinct from E. rhusiopathiae. However, the E. tonsillarum reference strain for serovar 14 (Iszap-4) and the reference strain for serovar 13 (Pecs-56), which has been said to represent a new genomic species, were found to cluster with typical isolates and reference strains of E. rhusiopathiae. Our reference strain for serovar 7 (Rotzunge) was also genetically typical of E. rhusiopathiae, thus indicating that these serotype reactivities cannot be relied upon as a means of identifying isolates as E. tonsillarum. Australian field isolates of E. rhusiopathiae were genetically diverse. Those recovered from sheep or birds were more diverse than those isolated from pigs, and isolates of serovar 1 were more diverse than those of serovar 2. The diversity found among isolates of the same serovar and the presence of isolates of different serovars in the same electrophoretic types (ETs) indicated that serotyping of E. rhusiopathiae was unreliable for use as an epidemiological tool. Some ETs contained isolates recovered from different animal species. ET 41 contained 32.2% of the field isolates and two reference strains, indicating that this clone of E. rhusiopathiae is both widespread and commonly associated with disease in various species of animals.

Animals↗

Cellular fatty acid composition of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum.

The cellular fatty acid compositions in 6 strains of Erysipelothrix rhusiopathiae and 7 strains of Erysipelothrix tonsillarum were determined by gas chromatography. Fatty acids, ranging from C10 to C18, were detected in the test strains. The fatty acid profile was characterized by very high percentages of 18:1 (cis-9) (cis-9-octadecenoic acid; 72.4 to 82.1%) and 16:1 (hexadecenoic acid; 8.7 to 13.7%). The profiles of the E. rhusiopathiae and E. tonsillarum strains resembled each other, indicating that discrimination between E. rhusiopathiae and E. tonsillarum from qualitative or quantitative fatty acid differences is difficult.

Animals↗

Enzymatic profiles of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillae.

The enzymatic activities of 39 strains of Erysipelothrix rhusiopathiae and 34 of E tonsillae were determined with the API ZYM system. The profiles of these two species were very similar, differing solely in N-acetyl-beta-glucosaminidase activity. Whereas 90 per cent of strains of E rhusiopathiae exhibited strong activity with N-acetyl-beta-glucosaminidase, positive reactions were observed for this enzyme in only 24 per cent of strains of E tonsillae. These results support previous DNA-DNA hybridisation studies and suggest that E tonsillae is a new species of the genus Erysipelothrix.

Animals↗

The detection and recovery of Erysipelothrix spp. in meat and abattoir samples in Western Australia.

AIMS: To investigate the occurrence of Erysipelothrix rhusiopathiae and other Erysipelothrix spp. in abattoir and meat samples in Western Australia. METHODS AND RESULTS: Samples were collected from various parts of pig and sheep carcasses, as well as different sections of slaughtering line, pen soil and effluent. Previously evaluated culture methods were applied for the isolation of Erysipelothrix spp., in conjunction with phenotypic and genotypic detection and identification procedures. Of 109 samples from the two abattoirs, 35 (32.1%) were Erysipelothrix genus-specific PCR-positive. These came from swabs of animal exterior surfaces and joints, slaughtering areas, pig pen soil and abattoir effluent. Four samples (3.7%) from sheep arthritic joints and pig abattoir effluent were also E. rhusiopathiae species-specific PCR-positive. Of 123 carcass washing samples, 12 (9.8%) were genus-specific PCR-positive, and these came from all five kinds of meat samples tested, including beef, lamb, mutton, pork and chicken. Four of them (3.3%) were also species-specific PCR-positive. A total of 25 isolates was recovered from the samples, of which seven were identified as E. rhusiopathiae, seven were consistent with E. tonsillarum, and the remaining 11 were other species of Erysipelothrix. CONCLUSIONS: Erysipelothrix spp. can still be isolated and identified from specimens of animal origin with relative ease, provided that appropriate cultural and molecular procedures are used. Clinical microbiology laboratories may need to improve their diagnostic protocols. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that E. rhusiopathiae and other species of Erysipelothrix continue to colonize and contaminate farmed animals and animal products. Erysipelothrix infection still poses a potential threat to the economy of the farmed animal industry, as well as being a potential human public health hazard.

Abattoirs↗

Comparison of methods for detection of Erysipelothrix spp. and their distribution in some Australasian seafoods.

For many years, Erysipelothrix rhusiopathiae has been known to be the causative agent of the occupationally related infection erysipeloid. A survey of the distribution of Erysipelothrix spp. in 19 Australasian seafoods was conducted, and methodologies for the detection of Erysipelothrix spp. were evaluated. Twenty-one Erysipelothrix spp. were isolated from 52 seafood parts. Primary isolation of Erysipelothrix spp. was most efficiently achieved with brain heart infusion broth enrichment followed by subculture onto a selective brain heart infusion agar containing kanamycin, neomycin, and vancomycin after 48 h of incubation. Selective tryptic soy broth, with 48 h of incubation, was the best culture method for the detection of Erysipelothrix spp. with PCR. PCR detection was 50% more sensitive than culture. E. rhusiopathiae was isolated from a variety of different fish, cephalopods, and crustaceans, including a Western rock lobster (Panulirus cygnus). There was no significant correlation between the origin of the seafoods tested and the distribution of E. rhusiopathiae. An organism indistinguishable from Erysipelothrix tonsillarum was isolated for the first time from an Australian oyster and a silver bream. Overall, Erysipelothrix spp. were widely distributed in Australasian seafoods, illustrating the potential for erysipeloid-like infections in fishermen.

Animals↗

Occurrence of Erysipelothrix spp. in broiler chickens at an abattoir.

From September 1995 to August 1996, 750 chickens from 66 farms sent to an abattoir in Nagano Prefecture, Japan, were examined for the presence of Erysipelothrix spp. Erysipelothrix spp. were isolated from 118 (15.7%) of 750 skin samples, 27 (7.3%) of 372 hypoderm samples, 12 (1.9%) of 630 throat samples, 106 (59.2%) of 179 feather samples, and none of 257 spleen samples. Of 66 farms, 55 farms (83.3%) sent Erysipelothrix-positive chickens and 11 farms (16.7%) only negative ones. Of 297 Erysipelothrix isolates, 273 isolates were identified as Erysipelothrix rhusiopathiae and 24 as Erysipelothrix tonsillarum. E. rhusiopathiae isolates were serotyped into nine different serovars. Of the 273 E. rhusiopathiae isolates, 33 (11.1%) were serotyped to serovar 6; 22 (7.4%) were serovar 5; 19 (6.4%) were serovar 2; 15 (5.1%) were serovar 8; 2 (0.7%) were serovar 21; 4 each (1.3% each) were serovars 1b, 9, 12, and 19; and 178 (59.9%) were untypeable. Of 24 E. tonsillarum isolates, 15 (5.1%) were serotyped to serovar 3, and 9 (3.0%) were serovar 7. These findings indicate that chickens seem to be a potential reservoir of Erysipelothrix spp. in nature and to be a source of human Erysipelothrix infection.

Animals↗

Erysipelothrix inopinata sp. nov., isolated in the course of sterile filtration of vegetable peptone broth, and description of Erysipelotrichaceae fam. nov.

Based on 16S rRNA gene sequence comparison, an isolate that was detected in sterile-filtered vegetable broth was classified as a novel member of the Erysipelothrix line of descent of the Firmicutes. Strain MF-EP02T resembles members of the two species of Erysipelothrix with validly published names, Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum, in morphology, fatty acid composition, lack of menaquinones in aerobically and anaerobically grown cultures, DNA G+C content and peptidoglycan amino acid composition. Distinct differences in physiological characteristics, however, support the allocation of this isolate to a novel species of the genus Erysipelothrix, for which the name Erysipelothrix inopinata sp. nov. (type strain, MF-EP02T=DSM 15511T=CIP 107935T) is proposed. Members of the Erysipelothrix line of descent are included in the family Erysipelotrichaceae fam. nov.

DNA, Ribosomal↗

Direct and rapid detection of Erysipelothrix rhusiopathiae DNA in animals by PCR.

Erysipelothrix rhusiopathiae is a gram-positive rod capable of causing erysipelas in swine. To establish a method for specifically detecting E. rhusiopathiae for practical applications, such as for the inspection of slaughterhouses, the feasibility of using primers derived from the DNA sequence coding for 16S rRNA in a PCR-specific detection system was investigated. Oligonucleotide primers were designed to amplify a 407-bp DNA fragment by PCR. The amplification was specific to the Erysipelothrix DNA but not to that of other bacterial genera tested. This PCR-based method efficiently and specifically detected the Erysipelothrix DNA sequence in joint and spleen samples from mice within 6 h, and application of the 407-bp DNA segment from samples containing very low numbers of bacteria (< 20 bacteria per spleen from mice) was possible. Although this PCR amplification is specific for the Erysipelothrix genus, which contains at least two species, E. rhusiopathiae and E. tonsillarum, it can be concluded that all Erysipelothrix strains detected by this PCR system in diseased pigs are E. rhusiopathiae because only E. rhusiopathiae is virulent for pigs. These results show that this PCR amplification system using the DNA sequence coding for 16S rRNA is very rapid and reliable and avoids cumbersome and lengthy cultivation steps, demonstrating that this system could be used for practical applications.

Animals↗